The ACVD task force on canine atopic dermatitis (XXIV): allergen-specific immunotherapy

Veterinary Immunology and Immunopathology 81 (2001) 363±383

The ACVD task force on canine atopic dermatitis (XXIV): allergen-speci®c immunotherapy Craig E. Grif®na,*, Andrew Hillierb b

a Animal Dermatology Clinic, San Diego, CA 92111, USA Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA

Abstract Allergen-speci®c immunotherapy (ASIT) has been used for years to treat dogs with atopic dermatitis (AD) and humans with atopic diseases. The ef®cacy of ASIT has been well documented for humans with respiratory atopic diseases and stinging insect allergy, but its effectiveness seems more controversial for patients with AD. In spite of insuf®cient evidence derived from randomized controlled trials, multiple open studies and a large body of clinical observations suggest that ASIT is effective in controlling the clinical signs of dogs with AD. As a result of the scarcity of evidence from controlled trials, the true ef®cacy of ASIT, and the optimal protocols for allergen dose and frequency of injection are currently unknown. Allergen-speci®c immunotherapy nevertheless may be included in the treatment of canine AD because of its potential advantages and limited disadvantages compared to other forms of therapy. There is no evidence, however, for the preference of any speci®c treatment protocol. The predictive value of historical, clinical and immunologic features related to the ef®cacy of ASIT in dogs with AD are discussed in this paper. Adverse reactions, and the requirements for monitoring of patients receiving ASIT, then are reviewed and detailed. Finally, this review highlights aspects of ASIT where further research and controlled studies are needed. # 2001 Published by Elsevier Science B.V. Keywords: Allergen-speci®c immunotherapy; Atopy; Atopic dermatitis; Hyposensitization; Dog

1. Introduction Allergen-speci®c immunotherapy (ASIT) is the practice of administering gradually increasing quantities of an allergen extract to an allergic subject to ameliorate the symptoms

*

Corresponding author. Tel.: ‡1-858-560-9393; fax: ‡1-858-560-0206. E-mail address: [email protected] (C.E. Griffin). 0165-2427/01/$ ± see front matter # 2001 Published by Elsevier Science B.V. PII: S 0 1 6 5 - 2 4 2 7 ( 0 1 ) 0 0 3 4 8 - 8

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associated with subsequent exposure to the causative allergen (WHO de®nition Ð Bousquet et al., 1998). Allergens are given in increasing doses up to a maintenance dose, or patient determined maximum dose. The terms desensitization, hyposensitization, and immunotherapy have all been used previously to describe this form of therapy. Desensitization and hyposensitization, the terms most commonly used for many years, refer to a reduction or loss of sensitivity (Anderson, 2000). These terms, nevertheless, do correlate with the clinical response seen in patients that undergo successful ASIT. This implies that this form of therapy lessens the patients' sensitivity, which is clinically the response we most commonly see. Unfortunately, this terminology does not re¯ect the mechanisms believed to be important, nor to the agents being used. More recently, the appellation immunotherapy was introduced. This name alone also is not appropriate as there are many forms of immunomodulating therapies that alter the immune response by stimulation and/or suppression. The designation allergen immunotherapy has been proposed by a WHO position paper (Bousquet et al., 1998). As the response is allergen-speci®c in humans (Lowell and Franklin, 1965; Norman and Lichtenstein, 1978), with similar ®ndings in dogs according to an unpublished report,1 this ACVD task force believes that the term allergen-speci®c immunotherapy is therefore most appropriate. Since the ®rst reports of ASIT for treatment of allergic rhinitis almost a century ago (Noon, 1911; Freeman, 1911), ASIT has been shown, in well controlled studies, to be an effective treatment for humans with allergic rhinitis, conjunctivitis or asthma and stinging insect hypersensitivity. The development of well-characterized and standardized allergens has further enhanced the therapeutic ef®cacy of ASIT in humans, with well-de®ned optimal maintenance doses of the major allergens having been established (Alvaraz-Cuesta et al., 1994). In dogs, ASIT was ®rst reported in the successful treatment of a dog with seasonal hay fever (Wittich, 1941). Over the past 30 years, a number of studies have reported on the ef®cacy of ASIT for canine AD, and this intervention is presently considered one of the mainstays of treatment for this disease. However, with rare exception, these studies have been designed as open and uncontrolled experiments with highly subjective variables for the assessment of clinical outcome. Furthermore, these studies have varied in their criteria for the diagnosis of AD, methodology for allergen selection, and treatment protocol employed. Thus, it is very dif®cult, if not impossible, to compare results of these studies and to draw any meaningful conclusions regarding the optimal indications for, and methods of, ASIT for treatment of canine AD. This paper will review ASIT in humans and dogs, and evaluate the evidence for the ef®cacy of ASIT in dogs with AD. It is not the intent of this paper to be a complete guide to ASIT for practitioners (for information on current ``standard protocols and recommendations'', the reader is referred to Reedy et al. (1997) or Scott et al. (2001)).

1 Anderson, R.K., Sousa, C.A., 1992. Workshop report 7. In vivo vs. in vitro testing for canine atopy. In: Ihrke, P.J., Mason, I., White, S. (Eds.), Advances in Veterinary Dermatology, Vol. II. Pergamon Press, New York, pp. 425±427.

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2. Mechanisms of action of ASIT 2.1. Human medicine Numerous changes in the immune reaction to allergen and the resultant in¯ammatory response have been documented in humans treated with ASIT (reviewed in Creticos, 1992; Nelson, 1998; Durham and Till, 1998; Akdis and Blaser, 2000; Theodoropoulos and Lockey, 2000). Brie¯y, these effects can be categorized as effects on the number of effector in¯ammatory cells and secretion of mediators (in particular, mast cells, basophils, and eosinophils); changes in concentration of allergen-speci®c immunoglobulins (slow decline of IgE, blunting of seasonal increase of IgE, and large increases in IgG1 and IgG4); and modulation of T-cell function and cytokine production (shift in favor of a type 1 response to allergen in place of type 2 response). Some of these changes, for example, the increase in allergen-speci®c IgG, appear to be useful markers of an immunologic response to ASIT, but do not necessarily re¯ect clinical improvement. The diminution of skin, nasal and conjunctival sensitivity to allergen exposure, have been reported to correlate with clinical improvement (Majchel et al., 1992; Nelson, 1998). A characteristic feature of ASIT is its ability to inhibit both immediate-phase and latephase responses after allergen challenge (Pienkowski et al., 1985; Fling et al., 1989; Iliopoulos et al., 1991; Varney et al., 1993; Malling and Weeke, 1993; Nelson, 1998). A review of studies of bee allergic patients treated with phospholipase A2 peptide immunotherapy (PLA-PIT) representing the immuno-dominant T-cell epitopes of this major bee venom allergen, has revealed interesting changes in T-cells and T-cell cytokine production in successfully treated patients (Akdis and Blaser, 2000). This review establishes that high allergen exposure in patients with high af®nity to speci®c allergenic epitopes results in higher density of MHC-II/allergen complexes on antigen presenting cells and stronger T-cell activation. Stronger activation favors gamma-interferon production and suppression of IgE secretion. Treated patients demonstrate allergen-speci®c anergy of peripheral T-cells that appears to correspond to increased IL-10 production. Moreover, ASIT induced IL-10 secretion stimulates allergen-speci®c IgG4 production, and decreases total and allergen-speci®c IgE. The type of antigen presenting cell also affects the T-cell response with B-cells and dendritic cells favoring a type 2 response while macrophages favor a type 1 response (Theodoropoulos and Lockey, 2000). 2.2. Canine medicine The mechanism of action of ASIT in canine AD is currently unknown. Similarly to the ®ndings in humans undergoing ASIT, one study reported signi®cant increases in allergenspeci®c IgG levels in dogs after at least 6 months of treatment (Hites and Kleinbeck, 1989). The authors of this study suggested that monitoring allergen-speci®c IgG would be helpful in assessing the bene®ts of ASIT, and thus could serve as a guideline to clinicians for maintenance or termination of treatment. However, an assessment of clinical improvement was not reported in this study and it has yet to be determined whether the reported increase in allergen-speci®c IgG was associated with a clinical response, or merely served as a marker of the immunologic response to ASIT.

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Another study reported a loss of immediate-phase intradermal allergen test reactions that paralleled the clinical improvement in dogs receiving ASIT (Willemse et al., 1984). In this report, 6 out of 16 dogs with AD whose signs improved with ASIT lost immediatephase skin test reactivity to relevant allergens. However, the timing of the intradermal test during treatment was not clearly stated, and furthermore, it was also reported that four out of ®ve dogs that improved with placebo (without allergens) also lost immediate-phase intradermal allergen test reactivity. These contradictory observations clearly warrant further evaluation. 3. Use of allergen-specific immunotherapy in human medicine Allergen-speci®c immunotherapy has been used for many years in human medicine, primarily for the treatment of allergic diseases caused by environmental and venom allergens. Multiple studies, and meta-analyses of controlled studies, have shown ASIT to be effective in stinging insect hypersensitivity, allergic rhinitis, allergic conjunctivitis, and allergic bronchitis. The ef®cacy of ASIT with standardized allergens of many tree/grass/ weed pollens, house dust mites, storage mites, molds, animal danders, and stinging insect venoms has been reported (Abramson et al., 1995; Bousquet et al., 1998; Nelson, 1998; Theodoropoulos and Lockey, 2000). It is important to note that most of these studies evaluated ASIT restricted to one or few allergens (i.e. in patients with one or few positive reactions on ``allergy testing''). One study that compared the ef®cacy of ASIT in patients allergic to grasses or multiple pollen species indicated that grass pollen allergic patients, but not the polysensitized patients, were signi®cantly improved after immunotherapy (Bousquet et al., 1991). However, some older studies do show response after mixed pollen ASIT (Lowell and Franklin, 1965). In human allergic patients, the response to ASIT appears to be allergen-speci®c (Lowell and Franklin, 1965; Norman and Lichtenstein, 1978). An improved ef®cacy is associated with several factors, that include higher doses of allergen, hypersensitivity to one or few allergens, the commencement of ASIT at a young age, and the onset of ASIT in patients with mild disease (prior to the development of chronic in¯ammatory changes of the affected organ) (Bousquet et al., 1988; Majchel et al., 1992; Haugaard et al., 1993). Although high dose therapy is reported to be more effective, risk/bene®t analysis suggests that intermediate doses may be preferable because of the lower occurrence of adverse reactions (Johnstone and Crump, 1961; Haugaard et al., 1993; Alvaraz-Cuesta et al., 1994). The advantages of ASIT early in the disease process include: prevention of chronic in¯ammatory changes, prevention of the further development of severe disease, prevention of the development of new hypersensitivities, and a possible reduction of adverse reactions due to a lower state of hypersensitivity (Malling and Weeke, 1993; Bousquet et al., 1998).2 It has also been suggested that ASIT may prevent the natural progression of further allergic disease in other organs (Mastrandrea, 2001). 2 DesRoches, A., Paradis, L., Knani, J., Hejjaoui, A., Dhivert, H., Michel, F.B., Bousquet, J., 1995. Specific immunotherapy prevents onset of new sensitization in monosensitized children. J. Allergy Clin. Immunol. 95, 309 (Abstract 674).

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3.1. Route of administration In humans, multiple routes of allergen administration have been assessed, although the majority of studies have evaluated subcutaneous injections of allergen. Ef®cacy with sublingual/oral ASIT has been demonstrated (Mastrandrea et al., 2000; Passalacqua et al., 1998). Intranasal (for allergic rhinitis) and intrabronchial (for asthma) ASIT have also been reported to reduce clinical signs by inducing a local immune response without evidence of a systemic response (Nelson, 1998). 3.2. Efficacy of ASIT in human patients with atopic dermatitis Recent textbook and review papers report that AD is not considered responsive to allergen immunotherapy, or that the few reported studies yield variable results or are inconclusive (Brehler et al., 1997; Nelson, 1998; Leung et al., 1999). However, several small uncontrolled studies and case reports have suggested ef®cacy of allergen immunotherapy in the treatment of AD (De Fuenmayer and Champion, 1979; Ring, 1982; Chait and Allkins, 1985; Zachariae et al., 1985). In fact, a recent review has questioned the interpretation that ASIT is ineffective for treatment of allergic signs in humans with AD (Mastrandrea, 2001). Two double-blind randomized placebo-controlled trials have been reported. One study was designed with at least a 2-year follow-up, ``tailor made'' mixed alum precipitated allergens to which the patients exhibited positive skin tests, and treatment with relatively low maintenance doses (0.4 ml of 1000 PNU/ml every 21 days). This study reported that signi®cantly more allergen-treated patients (13 out of 16, 81%) improved with ASIT compared to placebo-treated individuals (4 out of 10, 40%) (Kaufman and Roth, 1974). However, in light of more recent understanding of AD and ASIT, and methods for monitoring clinical response to therapy these results should be interpreted with caution. The low dose of allergen immunotherapy used in this study was markedly lower than intermediate to high doses used for treatment of asthma and allergic rhinitis. The low dose could be important as higher doses tend to be associated with higher response rates (Nelson, 1998). Another double-blind placebo-controlled study compared treatment with tyrosineadsorbed Dermatophagoides pteronyssinus allergen to placebo over a period of 8 months in children with severe atopic eczema (Glover and Atherton, 1992). The maintenance dose of the actively-treated group was the equivalent of 789 IU of Der pI (approximately one-®fth the quantity of allergen used for intradermal testing), administered monthly. There was no signi®cant difference in clinical signs, itch, or use of topical corticosteroids between groups of allergen-treated and placeboreceiving individuals. A small number of allergen-treated patients that were continued on ASIT for a further 6 months exhibited a greater improvement of clinical signs than those continued on placebo. However, although the authors note that it was not possible to conclude that allergen immunotherapy was ineffective, the very substantial placebo effect and the low power of the study did not allow any reasonable conclusions to be made. The low dose of allergen immunotherapy used in this study was different from the studies that showed ef®cacy with intermediate to high doses

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used in asthma and allergic rhinitis and therefore these results should also be interpreted with caution. Several other open studies published in the 1990s suggest some ef®cacy but, as most were uncontrolled, this must be carefully interpreted. A recent review discussed these publications and raised some concerns about the dif®culty of performing placebo-controlled blinded studies that require years to see the bene®t of active therapy and the high drop-out of patients among the placebo group (Mastrandrea, 2001). The relative dearth of well-controlled studies of conventional ASIT in the treatment of AD in humans is somewhat surprising. It would appear that the reportedly poor response to ASIT in AD is based on a very small body of evidence and it has yet to be established whether ASIT is, or is not, an effective mode of treatment in human AD. More recent studies have assessed various modi®cations of conventional subcutaneous ASIT in the treatment of AD. An open study with long-term follow-up of sublingual therapy in AD suggested this route is effective (Mastrandrea et al., 2000). Another study compared ASIT alone, gamma-interferon therapy, and combined gammainterferon/house dust mite ASIT with controls (Noh and Lee, 2000), and found that only the combination therapy improved the AD scores. The use of antigen/autologous antibody complexes for immunotherapy has also been demonstrated to be safe and effective in a double-blind placebo-controlled study of 24 patients with AD (Leroy et al., 1993). 4. Use of allergen-specific immunotherapy in dogs with atopic dermatitis 4.1. Indications for ASIT Allergen-speci®c immunotherapy is indicated in any dog where a diagnosis of atopic dermatitis has been made (see ACVD task force section XV, this issue), in which intradermal testing or allergen-speci®c IgE serology have enabled the identi®cation of allergens that are likely to be contributing to the disease and in which allergen contact is unavoidable. Moreover, the dog's owners should be ready to afford the time, expense and technical aspects of this regimen. In addition, when symptomatic anti-in¯ammatory therapy is ineffective, or associated with unacceptable or potentially unacceptable side effects (e.g. glucocorticoids), or is impractical to maintain for an extended period of time, then ASIT is indicated, even in dogs with seasonal disease of short duration (see ACVD task force section XIX, this issue). As ASIT is believed to alter the natural course of the allergic reaction, it is the only therapy that can prevent the development of further allergy. Indeed, ASIT offers the hope of long-term remission and can result in a therapy requiring a relatively low frequency of administration, especially compared to oral drugs that require administration daily or a few times a day. In contrast, it may be dif®cult to achieve and maintain long-term control of the clinical signs of AD with alternative forms of therapy such as allergen avoidance and symptomatic treatment. In addition, life threatening adverse reactions are rarely reported and there are no reports of long-term side effects from ASIT in the dog, thus few contra-indications would appear to exist for

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Table 1 Allergen immunotherapy: advantages and disadvantagesa Advantages

Disadvantages

Less frequent treatment administration than symptomatic therapy Less labor and time required, thus increased compliance No risk of long-term side effects reported Low risk short-term side effects Some dogs accept injections more readily than oral medications May permanently alter the course of the disease with possible cure Often more cost effective, especially in large breed dogs Preventative, not reactive treatment No monitoring tests required

Syringes and needles dispensed to client

a

Owner fear of giving injections Risk of anaphylaxis Only available in glass vials, breakage risk and cost Some dogs do not tolerate injections Client education and support required for efficacy Initially more expensive, with risk the expense will have no benefit

Modified from Griffin (1998).

the use of this therapy. A summary of the advantages and disadvantages of ASIT is provided in Table 1. The advantages of ASIT early in the disease process as outlined in humans (see Section 3 above), in particular, the prevention of the development of new hypersensitivities, have not been reported in the dog. As awareness of AD by veterinarians increases, it is likely that the disease will be diagnosed in younger dogs at early stages of disease development. Thus, studies of the natural progression of allergen hypersensitivity and AD and the effects of early ASIT are needed. 4.2. Variables of ASIT technique that can affect efficacy It is not our intention to provide a complete guide to ASIT. A review of the veterinary literature reveals that much of the accepted methodology regarding ASIT comes from anecdotal observations or open studies. Thus, it is extremely dif®cult at this time to draw ®rm conclusions regarding any of the variables involved with this treatment and comparison of studies is simply impossible (Table 2 outlines the variations in reported studies on ASIT for canine AD). Only one double-blind placebo-controlled study of the ef®cacy of ASIT in dogs with AD has been reported (Willemse et al., 1984). Besides the requirements for suitably blinded randomized controlled trials, there are two important problems that studies of ASIT in dogs must address. Firstly, allergen extracts should be standardized and the quantity of major allergen de®ned so that optimal, repeatable and reliable dosing schedules can be established. As the dose of allergen is critical to the success of allergen immunotherapy in humans (Bousquet et al., 1998), this is an important issue that needs to be controlled and evaluated in dogs with AD. As allergen sources are so variable and the recognition of relevant allergenic epitopes in dogs can differ from that in humans, this issue of comparing dose response rates will be dif®cult to accurately study. For a more detailed discussion of the variables regarding allergens (see ACVD task force section IV, this issue).

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Table 2 Variations in study designs and protocols in ASIT trials in dogs Variable

Examples

``Allergy testing'' methodology Intradermal testing Allergen-specific IgE serology Type of allergen

Aqueous Alum precipitated Propylene glycol suspended Glycerinated

Allergen source

Numerous allergen supply commercial companies

Induction protocols

Frequency of injections Rapidity of increasing allergen dose

Dose of allergen

Induction doses Maintenance dose Volume of allergen Number of allergens

Concentration of allergen

Protein nitrogen units (PNU) Weight/volume Noon units Biological units Per allergen in formula

Response criteria

Owner-based questionnaire Clinical criteria scored by clinicians with variable but limited scoring criteria Owner assessment based on recheck evaluation Concurrent medications allowed and/or lack of assessment for concurrent medication

Secondly, the clinical outcome should be documented using objective criteria. This is achieved in humans by either monitoring changes in symptoms, clinical signs and medication scores, or by provocative allergen challenges with objective measurement of end-organ (e.g. lung) function or mucosal (e.g. conjunctival, nasal, or bronchial) secretions at the challenged site (Malling and Weeke, 1993; Bousquet et al., 1998; Nelson, 1998). As no methods for controlled allergen challenge of the skin that induce clinical disease in the dog have been established, there is a need to develop a validated symptom and medication scoring system that can be easily and universally applied for use in future studies of canine AD. Thus, it becomes obvious that much needs to be done before we can determine which variables in¯uence the outcome of ASIT in the dog, and later de®ne the criteria and parameters for optimal results. These variables are outlined in Table 3 and further discussed below. 4.2.1. Allergen selection Clinical response to ASIT in dogs appears to be allergen-speci®c (see Footnote 1). In this workshop report, one of the participants (T. Willemse) reported the results of a

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Table 3 Variables of veterinary aqueous allergens in maintenance therapy Variable

Examples

Total protein concentration of maintenance ASIT

Typical moderate: 10,000±20,000 PNU/ml High strength: 20,000±40,000 PNU/ml Low strength: 1000±2000 PNU/ml

Protein concentration per allergen Typical: 1000±10,000 PNU/ml Allergen formula dose injected

Typical: 1 ml Range: 0.05±1 ml

Average allergen dose per day

High: 0.1 ml per day Low: 0.025 ml per day

Frequency of injection

Typical: every 21 days Frequent: every 3±7 days Infrequent: every 28±31 days Variable frequency: as needed and may change in different seasons

Number of allergens treated

Typical: 10 or less High: 12±24 often in multiple sets of formula but some will mix in one set Ultrahigh: 25±40

Mold allergens

Included or not included If included, in separate vial or mixed with other allergens

double-blind study, in which dogs treated with a nonspeci®c standard set of allergen exhibited a median improvement of clinical scores of 18%, while dogs treated with the allergens speci®cally selected on the basis of intradermal testing exhibited a median improvement of clinical scores of 70%. The allergens used in this study were alumprecipitated and both groups of dogs were treated for 8 months. Thus, careful consideration of the clinical signi®cance of positive reactions detected with intradermal testing or allergen-speci®c IgE serology, by evaluation of the history and likely exposure to allergens in each individual patient, appears to be important to yield better clinical outcome. 4.2.2. Allergens mixtures In humans, it is preferred that ASIT is prescribed either with a single allergen, or with mixtures of related cross-reacting allergens (e.g. related grass/tree/weed pollens or related mite species), or with mixtures of unrelated allergens provided that stability data are available (Bousquet et al., 1998). Two problems that may occur with allergen mixtures are an excessive dilution (ensuring suboptimal dosing and more rapid allergen deterioration) and loss of allergenicity as a result of the enzymatic activity of some allergen extracts. As the majority of dogs with AD exhibit multiple allergen hypersensitivity, mixtures of allergens are often prescribed. Studies of the effect of mold allergen extracts (known to be high in proteases) on weed, tree and grass pollen extracts by ELISA inhibition and skin test end-point titration testing, have demonstrated signi®cant loss of biologic activity of

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grass and weed pollens after these allergens are mixed and stored together (Rosenbaum et al., 1996).3 The clinical implications and potential effects on ASIT ef®cacy in relation to allergen mixtures have not been investigated. It should be noted that the ef®cacy reported in most veterinary studies is based on treatment with mixed allergens that often included molds. Studies to evaluate the stability of mixtures of unrelated allergens, the effect of storage and handling conditions on these mixtures, and whether the use of all ergen mixes affects ef®cacy of ASIT, are unfortunately lacking. 4.2.3. Route of administration Subcutaneous injections are the standard route of administration for ASIT in dogs. Other methods of allergen administration for AD in dogs have not been evaluated. One study initiated hyposensitization with intradermal injections in a rush protocol but changed to subcutaneous for maintenance (Mueller and Bettenay, 2001), but results of ef®cacy were not reported. Oral allergen treatment did induce tolerance in dogs experimentally sensitized to ovalbumin but not in two dogs sensitized to house dust mite antigen Der pI (Deplazes et al., 1995). 4.2.4. Number of allergens In the past, authors of textbooks have suggested, conventionally, that no more than 10± 12 allergens should be included in any single treatment set for ASIT. Early uncontrolled, open studies reported that dogs with 10 or less allergen hypersensitivities were more responsive to ASIT than dogs with more than 10 allergen hypersensitivities (Scott, 1981). A textbook published an open study that reported the highest clinical response in dogs treated with 11±20 allergens (Angarano and MacDonald, 1991). More recently, further uncontrolled open studies showed no association between the ef®cacy of ASIT and the number of allergens injected (Nuttall et al., 1998; Scott et al., 1993). However, we emphasize that, to date, there are no controlled studies that were performed to substantiate any of these observations. It could be speculated that the inclusion of high numbers of allergens in ASIT might not be important when one considers the conceptual hypothesis of the ``threshold effect'' (see ACVD task force section XIII, this issue). Animals that show hypersensitivity to multiple allergens theoretically might be successfully managed with ASIT that does not include all the allergens as long as a suf®cient number of clinically relevant allergens have been included in the treatment. However, until we know the true mechanism of how ASIT really works, one could also speculate that other mechanisms might explain favorable responses when only some of the allergens causing positive allergy test results are included in the treatment formula of multisensitized cases. 4.2.5. Dose of allergens In human medicine, the allergen dose for optimal clinical response has been de®ned for many standardized allergens (Bousquet et al., 1998). As previously noted, no suitably

3

Esch, R.E., 1992. The effect of fungal protease on the immunogenic activity of grass pollen extract. J. Clin. Immunol. 89, 149 (Abstract 17).

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standardized allergen extracts are available for use in veterinary medicine. In addition, authors of studies and textbooks have reported the concentration of allergens using variable schemes, such as Noon units, weight/volume or protein nitrogen units/ml (Willemse et al., 1984; Reedy et al., 1997; Scott et al., 2001). Further dif®culties arise when the concentration of each allergen in the ®nal mix is not clearly stated. Even though the ®nal total concentration of allergens usually is stated, there could be a variable number of allergens contributing to that total. For example, in the only double-blind placebo-controlled study of the ef®cacy of ASIT (Willemse et al., 1984), it is unclear whether the reported concentration of pollen allergen in each vial refers to all, or each of, the pollen allergens included. Thus, it becomes obvious that any meaningful comparison between studies employing different allergen doses is not possible. By convention or habit, maintenance vials with a total concentration of 10,000± 20,000 PNU/ml of allergens have been included in textbooks (Reedy et al., 1997; Scott et al., 2001). However, there are no studies that con®rm these concentrations to be any better or worse than any other concentration or dose of allergens. Reports of open studies demonstrating a higher response rates with high dose protocols4 the ef®cacy of low dose protocols,5 and the need for reduced allergen concentration for small dogs (Scott et al., 2001), remain to be veri®ed with appropriately controlled studies. One open study that compared the response rate in two groups of dogs receiving ASIT found a signi®cant difference with the standard ``high dose'' group 1 exhibiting 77% good-to-excellent response while the modi®ed ``low dose'' group two exhibited a 96% good-to-excellent response (Rosser, 1998). Dogs from group 2 received a standard protocol with 1 ml of 20,000 PNU/ml injected every 7±30 days as needed. Dogs were entered into group 2 because they had a persistent worsening of pruritus or an unacceptable adverse reaction. In this second group, dogs were given a maximum concentration of 10,000 PNU/ml and lower concentration were used in dogs still exhibiting pruritus and/or adverse reaction to even the 10,000 PNU/ml. In this study, the determination of pruritus, adverse reactions and assessment of improvement were based solely on the dog owners subjective observations reported to the investigator telephonically. Further studies are warranted to determine if the increased ef®cacy can be seen because of the lower dose or because highly sensitive dogs that had adverse reactions (including pruritus) exhibited a better response rate, and to determine whether similar results are obtained with more objective measures of clinical outcome. As some of these studies suggest the use of low doses of allergen for ASIT, consideration needs to be given to the stability of allergen dilutions. Allergen extracts in aqueous solution lose potency with time, with an increased loss of potency seen with increasing dilution (Nelson, 1981). For intradermal testing, diluted allergens can be kept refrigerated in glass vials for 8 weeks, and refrigerated in glass or plastic syringes for 2 weeks (see ACVD task force section IV, this issue). The potency of diluted allergens stored 4

Garfield, R.A., 1992. Injection immunotherapy in the treatment of canine atopic dermatitis: comparison of 3 protocols. In: Proceedings of the Annual Meeting of the American Academy of Veterinary Dermatology and American College of Veterinary Dermatology, Montreal, Canada, pp. 7±8. 5 Wagner, R., 1998. A retrospective survey of hyposensitization using low concentrations of alum-precipitated allergens. In: Proceedings of the 15th Annual Congress of the European Society of Veterinary Dermatology and European College of Veterinary Dermatology, Maastricht, The Netherlands, p. 167.

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for more than 2 months (as is often the case with allergen immunotherapy vials) needs to be evaluated further. 4.2.6. Frequency of aqueous allergen administration The interval between injections, during either initial loading or later maintenance phases of ASIT, has also not been evaluated in the dog. Once again, by convention or habit, textbook authors have suggested loading protocols with injection intervals of 2±3 days up to 1 week as the dose of aqueous allergen is increased, and maintenance protocols of between 5 and 20 days (Reedy et al., 1997; Scott et al., 2001). Two recent papers and an abstract reported results of rush protocols using increasing concentrations of allergen, administered intradermally or subcutaneously, every 20±30 min to reach a maintenance dose of 1.0 ml (20,000 PNU/ml) within 6 h (Patterson and Harris, 1999; Mueller and Bettenay, 2001).6 Such protocols were proposed to reduce the time required to reach the higher maintenance dose, and thus, possible time to maximal ef®cacy was reduced. Results of an open study suggested that a modi®ed protocol based on the patients' response to the injected allergens yielded a trend towards a decreased interval between maintenance injections (average 10 days, range 3±21 days) (Rosser, 1998). Others have reported that the ®nal interval between maintenance injections, in dogs whose signs improved, ranged from 3 weeks to 5 months (median: 2 months) (Willemse et al., 1984), or 1±12 months (Scott, 1981). As with the discussion of allergen concentration, the optimal dosing interval for both loading and maintenance allergen injections, has not been established in controlled studies, and can be highly variable between individual patients. The principal reason for a loading phase of increasing doses of allergen given with a short interval between injections is, supposedly, to reduce the incidence of severe life threatening adverse reactions while trying to achieve the high maintenance dose in the shortest period of time. The improved safety of one loading protocol over another has not been established. In the unpublished study of the rush protocol (see Footnote 6) by subcutaneous injection none of the 20 dogs (6 laboratory-bred dogs and 14 client-owned dogs with AD) exhibited any adverse reactions. However, in a recent study evaluating the safety of rush ASIT with intradermal injection of increasing concentrations of allergen every 30 min over 6 h, the rush protocol was discontinued prematurely in 8 out of 30 dogs (27%) due to increasingly severe pruritus in seven dogs and generalized wheal formation in one dog (Mueller and Bettenay, 2001). Whether this difference in rate of adverse reactions re¯ects the different route of allergen exposure or some other aspect is not known. As noted by the authors of both studies, such protocols require adequate preparation and close monitoring of each patient in a veterinary hospital. 4.3. Efficacy of ASIT in canine AD Results of numerous open uncontrolled studies have implied that ASIT is ef®cacious in the treatment of canine AD (Halliwell and Schwartzman, 1971; Halliwell, 1977; Nesbitt, 6 MacDonald, J.M., 1999. Rush hyposensitization in the treatment of canine atopy. In: Proceedings of the 15th Annual Meeting of the American Academy of Veterinary Dermatology and American College of Veterinary Dermatology, Maui, HI pp. 95±97.

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1978; Reedy, 1979; Scott, 1981; Sousa and Norton, 1990; Scott et al., 1993; Miller et al., 1993; Mueller and Bettenay, 1996; Schwartzman and Mathis, 1997; Nuttall et al., 1998; Rosser, 1998; Park et al., 2000)7,8,9,10,11 (see Footnotes 3±6). The results of these studies suggest that 50±100% of dogs receiving ASIT will exhibit at least a 50% improvement in their clinical signs after more than 4 months. There is only one paper reporting results of a double-blind placebo-controlled trial investigating the ef®cacy of ASIT in dogs with AD (Willemse et al., 1984). In this paper, it is stated that 16 out of 24 dogs (59%) treated with ASIT exhibited 51% decrease in clinical signs, compared to only 5 out of 24 dogs (21%) treated with placebo (Willemse et al., 1984). A complete remission of clinical was reported in 9 out of 16 dogs treated with ASIT and four out of ®ve dogs treated with placebo. However, a close review of the data raises several questions that should be considered in interpreting the results of this study. The timeframe as to when the response was determined and the data collected for statistical analysis were not presented clearly. This paper reported only a trend for signi®cance during the ®rst 12 months of the study, and signi®cant differences between groups were only apparent at time period 5 (i.e. 15 months). As shown in Fig. 1 in this paper, there is a very high drop out rate of dogs from both groups such as by 1 year (period 4) only 29 out of 51 dogs are still evaluated and after 18 months (period 6), only 10 out of 51 dogs remained enrolled in the study. Moreover, at the 12-month end-point, only scores of 16 of the original 24 dogs receiving placebo were depicted, and this decreases rapidly to ®ve dogs at the 15-month (period 5) and 18-month (period 6) re-evaluations and two dogs after 21 months (period 7). In this paper, the causes of drop out were not revealed, but it is conceivable that such a massive withdrawal of study subjects could have considerably biased the statistical analyses. Indeed, at the re-evaluation made during period 5 (12±15 months), in which variations in clinical scores are reported to be signi®cantly different between groups, there are only 11 dogs left in the active group (initial enrollment: 27 dogs) and ®ve dogs remaining in the placebo one (initial enrollment: 24 dogs). In this paper, and because of the very high drop out rate, interpretation of the data should have included intent-to-treat analyses. Finally, the criteria for inclusion of study subjects were not speci®ed clearly. This study employed alum-precipitated allergens (Al[OH]3 3 mg/ml) as adjuvant. Alum is used as a carrier to retard absorption of allergen (Holzman and Norman, 1966), and is believed to increase antigenic potency (Freund, 1947). In humans, numerous open 7 Griffin, C.E., Rosenkrantz, W.S., 1991. A comparison of hyposensitization results in dogs based on an intradermal protocol versus an in vitro protocol. In: Proceedings of the Annual Meeting of the American Academy of Veterinary Allergy, Scottsdale, AZ, p. 12. 8 Willemse, T., 1994. Hyposensitization of dogs with atopic dermatitis based on the results of in vivo and in vitro (IgGd ELISA) diagnostic tests. In: Proceedings of the Annual Meeting of the American Academy of Veterinary Dermatology and American College of Veterinary Dermatology, Charleston, SC, p. 61. 9 Griffin, C.E., Rosenkrantz, W.S., 1996. In vivo versus in vitro testing for atopy. Concurrent session notes. In: Proceedings of the Annual Meeting of the American Academy of Veterinary Dermatology and American College of Veterinary Dermatology, Las Vegas, NV, pp. 78±79. 10 Power, H.T., 2000. Why do owners discontinue immunotherapy? Vet. Dermatol. 11 (1), 14. 11 Scott, K.A., Rosychuk, R.A.W., White, S.D., 1999. Hyposensitization: the Colorado State University experience with emphasis on efficacy by breed. In: Proceedings of the Annual Meeting of the American Academy of Veterinary Dermatology and American College of Veterinary Dermatology, Maui, HI, pp. 107±109.

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studies have reported the clinical ef®cacy of alum-precipitated allergen ASIT for respiratory allergies (Fuchs and Strauss, 1959; Tuft et al., 1965; Tuft and Torsney, 1965; Frankland and Noelpp, 1966). In contrast, a report of a more recent study showed poor clinical response to alum-precipitated allergens (Bousquet et al., 1985), and other studies have shown a lack of alteration of immune response to allergen following alumprecipitated ASIT (Lichtenstein et al., 1968; Bousquet et al., 1985). Thus, it is possible that alum precipitation could affect the immune response to allergens, although the mechanisms of such effects remain unclear. As double-blind placebo-controlled studies using aqueous nonadjuvanted allergens for ASIT (as is the practice in the USA) have not been reported the ®ndings of the study by Willemse et al. (1984) need to be reproduced with aqueous allergens. Nevertheless, in spite of the lack of evidence based on randomized controlled trials, results of open uncontrolled studies, and a wide body of clinical observations by veterinary dermatologists throughout the world, suggest the likely clinical ef®cacy of aqueous ASIT for treatment of clinical signs in dogs with AD. Hopefully, future studies will document such ef®cacy in blinded randomized controlled trials. Unfortunately, the performance of these experimental trials is fraught with problems related to the long duration that seems required to document treatment ef®cacy, and by the multiple confounding factors and secondary diseases that are seen during the management of canine AD (see ACVD task force section XIV, this issue). Additionally, the use of placebo for such a long trial could be unethical and also lead to a very high rate of drop out, as seen in Willemse's trial (Willemse et al., 1984). Therefore, future studies may be more appropriate if designed as double-blind randomized trials in which placebo or ASIT are added to standard-of-care drugs. Treatment and medication scores will need to be closely monitored in addition to clinical scores for lesions and pruritus to determine ef®cacy of ASIT. Numerous reports of the ef®cacy of ASIT where allergen selection was based on the results of allergen-speci®c IgE serology have suggested that results are similar to those in which allergen selection is based on the results of intradermal testing (Sousa and Norton, 1990; Scott et al., 1993; Miller et al., 1993; Schwartzman and Mathis, 1997; Park et al., 2000) (see Footnotes 7±9). Furthermore, two reports (one unpublished and the other a personal observation reported at a workshop) suggest the ef®cacy of ASIT can be improved by selecting allergens based on the results of both allergen-speci®c serology (IgE in one report and IgGd in the other) and intradermal testing (see Footnotes 3 and 8). These are all open and uncontrolled studies and anecdotal observations, and the conclusions need to be validated in controlled studies before they can be accepted. In particular, the observation of similar ef®cacy of ASIT based on allergen-speci®c IgE serology compared to intradermal testing, cannot be cited as validation of the relevance of either ``allergy testing'' methodology until suitable controlled studies of ASIT are performed. 4.3.1. Time to efficacy of ASIT One paper reported that some dogs exhibit a favorable response to ASIT within the ®rst 2 months of treatment (Scott et al., 1993). Certainly some dogs can respond relatively rapidly but how common this phenomenon is has not been investigated speci®cally. In

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the trial, in which alum-precipitated allergens were utilized, at least 2 out of 24 dogs apparently responded very well to ASIT by 3 months, but no evaluation was done prior to 3 months, therefore it is impossible to determine how soon these dogs had really responded (Willemse et al., 1984). This study also determined that the evaluation of response to ASIT could be restricted to 9 months as dogs not responding by that time had a very poor chance of exhibiting a favorable response later on (Willemse et al., 1984). Whether this is true with ASIT employing aqueous allergens is unknown. Indeed, other papers analyzing data of ASIT with aqueous allergens reported that most dogs will respond from 3 to 12 months of treatment (Scott et al., 1993; Mueller and Bettenay, 1996). The time to clinical bene®t and total duration of ASIT before determination of treatment ef®cacy, or lack thereof, are currently unknown and clearly deserve further study. 4.3.2. Duration of ASIT The long-term ef®cacy of ASIT (i.e. persistence of bene®cial effects after discontinuation of injections) in dogs with AD has never been evaluated in a controlled study. Controlled studies of grass and ragweed pollen allergic human patients have shown longterm ef®cacy for several years following discontinuation of ASIT. Other studies with house dust mite and animal dander allergens suggest that ASIT should continue for at least 3 years if long-term ef®cacy is to be appreciated in at least some patients (Hedlin et al., 1991; Bousquet et al., 1998). In the dog, results of open studies with unspeci®ed or variable follow-up periods, have shown that ASIT could be discontinued in some patients, and that sustained bene®t was appreciated in 4% (4 out of 100) (Rosser et al., 1998), 6% (1 out of 17) (Park et al., 2000), 12% (6 out of 50) (see Footnote 7), and 23±35% (see Footnote 10) of dogs. Such variable and con¯icting data need to be re-evaluated in further controlled trials. Until this information becomes available, the clinician should expect that this form of treatment could be life long in some dogs, and that, in other patients, complete remission of clinical signs of AD also is possible. 4.3.3. Predictive factors for effective immunotherapy Many of the previously cited studies have reported that the ef®cacy of ASIT in dogs with AD can be variably affected by the following factors: the age at onset of disease (Nesbitt, 1978; Scott et al., 1993; Willemse et al., 1984); the age at time of commencement of ASIT (Scott et al., 1993); the duration of disease (Scott et al., 1993; Mueller et al., 1996; Nuttall et al., 1998); the severity of clinical signs (Grif®n, 1998), the breed (see Footnotes 8 and 11); the strength of intradermal test reaction (Reedy, 1979; Mueller et al., 1996); the number of intradermal test reactions (Schwartzman and Mathis, 1997); and the type of allergen to which the patient is hypersensitive (Reedy, 1979; Mueller et al., 1996; Schwartzman and Mathis, 1997).12 As previously mentioned, these reports can be regarded as no more than observations at present and these data need further validation in controlled studies. 12 Charach, M., 1993. Specific immunotherapy in dogs with house dust mite allergy: a double-blind placebo study. In: Proceedings of the Annual Meeting of the American Academy of Veterinary Dermatology and American College of Veterinary Dermatology, San Diego, CA, p. 50.

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4.3.4. Effects of glucocorticoids Glucocorticoids exhibit multiple, and often signi®cant, effects on the immune system, targeting mononuclear leukocytes especially, and lymphocytes, in particular (see ACVD task force section XX, this issue). The effects of concurrent glucocorticoid use during ASIT have not been evaluated in the dog, but such interaction could be important given the purported importance of T-lymphocytes in successful ASIT. It has been stated in textbooks that ASIT may still be effective if oral prednisone/ prednisolone doses are kept as low as possible or if they are administered on an alternate day basis (Scott et al., 2001). In contrast, authors of another textbook suggest that glucocorticoids should be avoided early in the induction phases of ASIT (Grif®n et al., 1993). One open study reported the use of alternate day prednisolone therapy in 15 out of 28 dogs during the induction phase of ASIT, with no apparent loss ef®cacy (Scott, 1981). These observations are not surprising, but need to be supported by controlled studies. Besides the concerns for the effects of glucocorticoids on the immune response to ASIT, it is theoretically possible that glucocorticoids could mask a positive clinical bene®t of ASIT, or more seriously, could mask covert adverse reactions needing a modi®cation of the treatment protocol. 4.4. Re-evaluation of patients during immunotherapy Various textbooks (Grif®n, 1993; Reedy et al., 1997; Scott et al., 2001), an open study (Rosser, 1998) and an unpublished study (see Footnote 10), have suggested that ongoing active follow-up of each patient is an important factor in the potential success of ASIT. Unlike most traditional forms of drug therapy, where extensive research of the pharmacokinetics and pharmacodynamics of a therapeutic agent facilitate the recommendation of speci®c dosing schedules, it would seem likely that the ef®cacy of ASIT in dogs could be improved by careful monitoring of the response in the individual patient for the following reasons: allergen extracts are poorly standardized and true biological potency may vary from batch to batch; canine patients exhibit considerably variable size and weight; the time until clinical improvement is detected could be months or even years; allergen immunotherapy could exacerbate the clinical signs of AD; and some dogs appear to exhibit only transient improvement following each injection. Furthermore, as reviewed elsewhere (see ACVD task force sections X±XII, this issue), canine AD is a complex disease where clinical signs maybe triggered by multiple factors with many dogs having signi®cant concurrent skin disease (for example, infections with Staphylococcus bacteria and Malassezia yeast, other concurrent allergic diseases). It is likely that many dogs that have been observed to fail to respond to ASIT would have shown signi®cant clinical improvement if these ¯are factors had been recognized and controlled through regular re-evaluations. Finally, compliance with ASIT by owners of dogs with AD appears to be low (Rosser, 1998) (see Footnote 10), as it is in humans (Bousquet et al., 1998). It is logical that frequent communication with the owners and re-evaluation of the patients is likely to improve compliance with ASIT for a suf®cient duration of time to assess possible clinical response.

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Thus, we believe that patient monitoring is essential, and a frequently under-appreciated aspect of ASIT. 4.5. Safety/adverse reactions Adverse reactions to ASIT are reported as uncommon to rare in dogs. One paper reported an incidence of 5% (Angarano and MacDonald, 1991). In another blinded placebo-controlled study, 3 of the 27 dogs treated with alum-precipitated allergens exhibited severe generalized pruritus while 2 of the 24 alum-containing placebo-treated dogs suffered from generalized pruritus, one of them associated with generalized urticaria (Willemse et al., 1984). In another study of 100 dogs undergoing ASIT, one suffered from anaphylaxis, ®ve had facial angioedema, and seven other dogs had milder adverse reactions though another 25 had a perceived worsening of the pruritus (Rosser, 1998). In this study, 38% of 100 dogs that began ASIT had adverse reactions (13%) or increased pruritus (25%) that were considered severe enough to require a change or discontinuation of therapy. Adverse reactions can be divided into local and systemic. 4.5.1. Local adverse reactions In one textbook, it is reported that small local reactions with swelling and/or erythema, are relatively common (Reedy et al., 1997), while pronounced local reactions with edema and sometimes pain or pruritus, are occasionally seen. However, there are no numerical data to support this statement. 4.5.2. Systemic adverse reactions Systemic reactions that have been reported include weakness, depression, anxiety or sleepiness, panting or hyperactivity, diarrhea, vomiting, increased bowel sounds or frequent swallowing, urticaria/angioedema, collapse or anaphylaxis and pruritus (Grif®n, 1998; Reedy et al., 1997; Rosser, 1998; Scott et al., 2001). The most common adverse reaction is a worsening of the clinical signs and pruritus. This may occur immediately after an injection or up to 1 or 2 days later, and can persist for hours to days. One report suggests that as many as 25% of dogs are persistently worse with immunotherapy (Rosser, 1998). More adverse reactions to house dust, house dust mite and mold allergens anecdotally have been reported but these are unsubstantiated (Bevier, 1990). The factors that may in¯uence the development of adverse reactions have not been well studied. In humans pretreatment with antihistamines can prevent these reactions (Nielsen et al., 1996) and this has been anecdotally recommended for dogs in a textbook (Grif®n, 1993). 5. Conclusions Despite the absence of suitably controlled studies to evaluate the ef®cacy of ASIT using aqueous allergens, the results of a double-blind placebo-controlled study with alumprecipitated allergens, as well as the overwhelming clinical experience of veterinary dermatologists world wide, provide observational evidence of the bene®ts of this therapy in the treatment of canine AD. There is a an urgent need for further research to clarify:

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1. The ``true'' efficacy of ASIT for the treatment of canine AD using aqueous allergen extracts (what proportion of dogs with AD are likely to respond to ASIT, and how much improvement can be expected in these patients). 2. Historical, clinical, and immunologic features of AD that may be predictive of response to ASIT. 3. Indications for alteration of ASIT protocol (allergen selection, allergen dose, frequency of injection, and other variables) for optimal efficacy. 4. Effects of concurrent glucocorticoids and other immunomodulating therapies on the response to ASIT. 5. The influence of early detection of AD and commencement of ASIT on the course of development of hypersensitivity to other allergens. In conclusion, ASIT is indicated for use in dogs with AD after careful selection of allergens for inclusion into the therapy, with regular and diligent follow-up evaluations, and in conjunction with other treatment modalities (see ACVD task force sections XIX±XXIV, this issue).

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