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**26**

Hak Sun Yu

*South Korea*

**Parasite-Derived Proteins Inhibit**

The prevalence of allergic disease and asthma has increased dramatically during the last 30-40 years. Atopic disorders comprise a range of allergic diseases including asthma, anaphylaxis, allergic rhinitis, and atopic dermatitis; these diseases have been seen a precipitous increase in the last four decades. Intriguingly, geographic regions with a high helminth infection burden tend to have a lower incidence of asthma (1). The effects of parasitic infections on the incidence of allergic disease has been receiving increased attention from researchers of late, with studies conducted in Ethiopia and Gabon demonstrating that parasitic infestation is associated with reduced atopic sensitization and dust mite skin test sensitivity (2-4). Children treated repeatedly for *Trichuris trichiura* and *Ascaris lumbricoides* exhibited increased dust-mite skin responses as compared with children that had not been treated for asymptomatic soilassociated helminthic infections (5). Several molecules from helminthes induce pronounced Th2 responses in a manner similar to that seen in cases of full-blown parasitic infection. Excretory-secretory (ES) glycoproteins isolated from the rodent nematode, *Nippostrongylus brasiliensis,* have been shown to evidence Th2-promoting activity on dendrite cells, however, the exact nature of the molecules involved in *N. brasiliensis* ES proteins remain to be clearly elucidated. This activity is heat-labile and protease-sensitive, thereby suggesting that the active component is proteinaceous in nature (6). Also, in schistosomiasis, the soluble extract of *Schistosoma mansoni* eggs (SEA) was shown to induce SEA-specific Th2 responses when injected into mice (7), and SEA was also demonstrated to condition human dendrite cells (DCs) to polarize Th response in a Th2 direction *in vitro* (8). When exposed to Th2 cytokines, these molecules can also activate host CD4+CD25+Foxp3+ T cells (regulatory T cells, Treg) which subsequently release IL-10 and tumor growth factor β (TGF-β), which may be functionally involved in the suppression of the level of Th2 cytokines IL-4, IL-5, and IL-13. These parasites can establish a chronic infection, which highlights important issues (9), in that the presence of these metabolically active pathogens indicates that the immune system is being relentlessly challenged with foreign antigens; this continuous immune reactivity, if uncontrolled, could eventuate severe pathology. In addition, these pathogens may have developed evolutionary strategies by which they may evade the immune system for long-term

In order to ascertain, then, whether parasitic infections can reduce allergic reactions and whether their infective stage influences the immune system of the host, we have mimicked

**1. Introduction**

survival in an immunocompetent host (10).

**Allergic Specific Th2 Response**

*Department of Parasitology, School of Medicine,*

*Pusan National University, Yangsan-si,*

### **Parasite-Derived Proteins Inhibit Allergic Specific Th2 Response**

#### Hak Sun Yu

*Department of Parasitology, School of Medicine, Pusan National University, Yangsan-si, South Korea*

#### **1. Introduction**

530 Allergic Diseases – Highlights in the Clinic, Mechanisms and Treatment

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expressed by blood-borne microfilariae of the parasitic nematode Brugia malayi inhibits human neutrophil serine proteinase. *Blood*, Vol.94, No.4, (August 1999), The prevalence of allergic disease and asthma has increased dramatically during the last 30-40 years. Atopic disorders comprise a range of allergic diseases including asthma, anaphylaxis, allergic rhinitis, and atopic dermatitis; these diseases have been seen a precipitous increase in the last four decades. Intriguingly, geographic regions with a high helminth infection burden tend to have a lower incidence of asthma (1). The effects of parasitic infections on the incidence of allergic disease has been receiving increased attention from researchers of late, with studies conducted in Ethiopia and Gabon demonstrating that parasitic infestation is associated with reduced atopic sensitization and dust mite skin test sensitivity (2-4). Children treated repeatedly for *Trichuris trichiura* and *Ascaris lumbricoides* exhibited increased dust-mite skin responses as compared with children that had not been treated for asymptomatic soilassociated helminthic infections (5). Several molecules from helminthes induce pronounced Th2 responses in a manner similar to that seen in cases of full-blown parasitic infection. Excretory-secretory (ES) glycoproteins isolated from the rodent nematode, *Nippostrongylus brasiliensis,* have been shown to evidence Th2-promoting activity on dendrite cells, however, the exact nature of the molecules involved in *N. brasiliensis* ES proteins remain to be clearly elucidated. This activity is heat-labile and protease-sensitive, thereby suggesting that the active component is proteinaceous in nature (6). Also, in schistosomiasis, the soluble extract of *Schistosoma mansoni* eggs (SEA) was shown to induce SEA-specific Th2 responses when injected into mice (7), and SEA was also demonstrated to condition human dendrite cells (DCs) to polarize Th response in a Th2 direction *in vitro* (8). When exposed to Th2 cytokines, these molecules can also activate host CD4+CD25+Foxp3+ T cells (regulatory T cells, Treg) which subsequently release IL-10 and tumor growth factor β (TGF-β), which may be functionally involved in the suppression of the level of Th2 cytokines IL-4, IL-5, and IL-13. These parasites can establish a chronic infection, which highlights important issues (9), in that the presence of these metabolically active pathogens indicates that the immune system is being relentlessly challenged with foreign antigens; this continuous immune reactivity, if uncontrolled, could eventuate severe pathology. In addition, these pathogens may have developed evolutionary strategies by which they may evade the immune system for long-term survival in an immunocompetent host (10).

In order to ascertain, then, whether parasitic infections can reduce allergic reactions and whether their infective stage influences the immune system of the host, we have mimicked

Parasite-Derived Proteins Inhibit Allergic Specific Th2 Response 533

inflammation response as the result of simultaneous OVA challenge, as compared to the immunization method. In particular, the Tl-ES treatment with OVA challenge group exhibited more severe lung inflammation than was observed in the immunized group. We believe that certain allergens or proteases might be included in the ES proteins, and parasitic proteases have also been identified as allergens (20-23). Sokol *et al*. previously suggested that a host-derived sensor of proteolytic activity might involve cleavage via parasite or allergic proteases. This sensor, once cleaved, activates the cells of the innate immune system

Fig. 1. **Histological findings of airway inflammation in ova-challenged control mice and the effect of immunization and challenge with OVA groups.** Female and 6 weeks of age mice were induced airway allergic reaction using intraperitoneally (I.P.) sensitizing with 75 ug of ovalbumin (OVA, Sigma, Grade V) and 2 mg of aluminium hydroxide gel, on days 0 and 7. One week after the final sensitization, the mice were intra nasal challenged with 50 ug of OVA on 4 consecutive days (days 13, 14, 19, and 20). We mentioned "Immunized group" that were injected by I.P. with 100 ul of 10 ug/ml *Toxascaris leonina* excretory-secretory (Tl-ES) and *T. leonina* total protein (Tl-TP), respectively, on 7 and 14 days before airway allergic reaction procedure. Also "Challenged with OVA" group were mentioned which were injected by I.P. with 100 ul of 10 ug/ml of Tl-ES and Tl-TP respectively at the same challenge days in airway allergic reaction procedure. The negative control group was challenged with PBS (I.N.) on the same challenge day in airway allergic reaction procedure. A; PBS-treated, B-F; OVA+ alumtreated (induced asthma), (C) immunized Tl-ES protein, (D) immunized Tl-TP, (E) Tl-ES

IL-4 has been demonstrated to regulate isotype class switching in B cells to IgE synthesis, and IL-5 stimulates eosinophil growth, activates these cells, and prolongs eosinophil survival. *T. leonina-*derived proteins could inhibit increases in the levels of IL-4 and IL-5 from OVA challenge. In particular, the level of IL-4 in the BAL fluid in the immunized group was almost half of that observed in the OVA-only challenge group. This result was consistent with the results regarding the IgE concentration in the blood (15). Although levels of the IL-5 cytokine of *T. leonina* ES and total protein-immunized mice were lower than those observed in asthma

protein treatment with OVA challenge, (F) Tl-TP protein with OVA challenge.

to induce a Th2 response (13).

chronic infection conditions in our experiment via treatment with parasite-derived proteins for one month prior to allergen treatment. We attempted to determine whether or not these parasite-derived proteins suppressed allergy-specific Th2 reactions.

#### **2. Immunization of parasite derived proteins inhibits allergic specific Th2 response**

Long-lived parasites are highly accomplished practitioners of immune evasion and manipulation, utilizing strategies honed during their long co-evolutionary interaction with the mammalian immune system (10, 11). How is the host affected by these parasitic strategies? Although many hypotheses have been advanced, until the present time there has been little evidence to support these theories. Th2 host response as the result of parasitic infection was apparent, although this is the result of parasitic strategies to escape from the host immune system or the result of a human protective system associated with parasitic infection. Additionally, we could readily detect Th2 responses in allergic patients. Thus, until now, there has been considerable controversy regarding the relationship between parasites and allergic reactions; particularly, whether parasites can evoke some allergicspecific response or can reduce allergen-specific responses (12-14).

We have attempted to determine whether parasite-derived proteins can accelerate or reduce asthma symptoms in accordance with the treatment period (15). We have mimicked chronic infection conditions in our experiment via treatment with parasite (*Toxascaris leonina* adult worm)-derived proteins [ES protein (Tl-ES) and total proteins (Tl-TP)] for one month prior to the administration of allergen treatment. We mentioned this group as Immunized group; also Challenged with OVA group were mentioned which were administrated Tl-ES and Tl-TP at the same challenge days in airway allergic reaction procedure. As results, the immunized Tl-ES and Tl-TP groups evidenced a thinning of the bronchial epithelial and muscle layer, a disruption and shedding of the epithelial cells, a reduction in the number of goblet cells as compared to the OVA-challenged groups (Fig. 1). When the airway functions of these mice were monitored, we detected that the Penh values by methacholine treatment (from 2.5 mg/ml to 25 mg/ml) were significantly higher in the OVA-inhalation mice than those of Tl-TP and Tl-ES immunized group (Fig. 2A). The numbers of most inflammatory cells (macrophages, eosinophils, lymphocytes and neutrophils) in the BAL fluids were significantly increased in all of the OVA-challenged groups (Fig. 2B & 2C). The administration of Tl-ES and Tl-TP prior to asthma induction (immunized group) and the Tl-ES and Tl-TP with OVA challenge (challenged with OVA groups) evidenced inhibited recruitment of inflammatory cells into the airway (Fig. 2B & 2C). In particular, neutrophils and lymphocytes were significantly reduced by the parasite proteins at any administration time (p value < 0.05). The total number of eosinophils of the immunized and OVAchallenged group were slightly reduced; however, this reduction was not statistically significant (P value > 0.05).

Although some articles have previously asserted that nematodes can induce allergic reactions during their larval migration period (16, 17), many articles have reported that parasitic infections, particularly chronic parasitism, help to reduce host allergic reactions and to modulate host immune responses (14, 18, 19). We have determined that immunization with *T. leonina* adult worm ES and total proteins induces a down-regulation of asthma-associated cytokines, including IL-4 and IL-5, in the bronchial alveolar lavage (BAL) fluids (Fig. 3). However, these proteins did not significantly influence allergic airway

chronic infection conditions in our experiment via treatment with parasite-derived proteins for one month prior to allergen treatment. We attempted to determine whether or not these

Long-lived parasites are highly accomplished practitioners of immune evasion and manipulation, utilizing strategies honed during their long co-evolutionary interaction with the mammalian immune system (10, 11). How is the host affected by these parasitic strategies? Although many hypotheses have been advanced, until the present time there has been little evidence to support these theories. Th2 host response as the result of parasitic infection was apparent, although this is the result of parasitic strategies to escape from the host immune system or the result of a human protective system associated with parasitic infection. Additionally, we could readily detect Th2 responses in allergic patients. Thus, until now, there has been considerable controversy regarding the relationship between parasites and allergic reactions; particularly, whether parasites can evoke some allergic-

We have attempted to determine whether parasite-derived proteins can accelerate or reduce asthma symptoms in accordance with the treatment period (15). We have mimicked chronic infection conditions in our experiment via treatment with parasite (*Toxascaris leonina* adult worm)-derived proteins [ES protein (Tl-ES) and total proteins (Tl-TP)] for one month prior to the administration of allergen treatment. We mentioned this group as Immunized group; also Challenged with OVA group were mentioned which were administrated Tl-ES and Tl-TP at the same challenge days in airway allergic reaction procedure. As results, the immunized Tl-ES and Tl-TP groups evidenced a thinning of the bronchial epithelial and muscle layer, a disruption and shedding of the epithelial cells, a reduction in the number of goblet cells as compared to the OVA-challenged groups (Fig. 1). When the airway functions of these mice were monitored, we detected that the Penh values by methacholine treatment (from 2.5 mg/ml to 25 mg/ml) were significantly higher in the OVA-inhalation mice than those of Tl-TP and Tl-ES immunized group (Fig. 2A). The numbers of most inflammatory cells (macrophages, eosinophils, lymphocytes and neutrophils) in the BAL fluids were significantly increased in all of the OVA-challenged groups (Fig. 2B & 2C). The administration of Tl-ES and Tl-TP prior to asthma induction (immunized group) and the Tl-ES and Tl-TP with OVA challenge (challenged with OVA groups) evidenced inhibited recruitment of inflammatory cells into the airway (Fig. 2B & 2C). In particular, neutrophils and lymphocytes were significantly reduced by the parasite proteins at any administration time (p value < 0.05). The total number of eosinophils of the immunized and OVAchallenged group were slightly reduced; however, this reduction was not statistically

Although some articles have previously asserted that nematodes can induce allergic reactions during their larval migration period (16, 17), many articles have reported that parasitic infections, particularly chronic parasitism, help to reduce host allergic reactions and to modulate host immune responses (14, 18, 19). We have determined that immunization with *T. leonina* adult worm ES and total proteins induces a down-regulation of asthma-associated cytokines, including IL-4 and IL-5, in the bronchial alveolar lavage (BAL) fluids (Fig. 3). However, these proteins did not significantly influence allergic airway

**2. Immunization of parasite derived proteins inhibits allergic specific Th2**

parasite-derived proteins suppressed allergy-specific Th2 reactions.

specific response or can reduce allergen-specific responses (12-14).

**response**

significant (P value > 0.05).

inflammation response as the result of simultaneous OVA challenge, as compared to the immunization method. In particular, the Tl-ES treatment with OVA challenge group exhibited more severe lung inflammation than was observed in the immunized group. We believe that certain allergens or proteases might be included in the ES proteins, and parasitic proteases have also been identified as allergens (20-23). Sokol *et al*. previously suggested that a host-derived sensor of proteolytic activity might involve cleavage via parasite or allergic proteases. This sensor, once cleaved, activates the cells of the innate immune system to induce a Th2 response (13).

Fig. 1. **Histological findings of airway inflammation in ova-challenged control mice and the effect of immunization and challenge with OVA groups.** Female and 6 weeks of age mice were induced airway allergic reaction using intraperitoneally (I.P.) sensitizing with 75 ug of ovalbumin (OVA, Sigma, Grade V) and 2 mg of aluminium hydroxide gel, on days 0 and 7. One week after the final sensitization, the mice were intra nasal challenged with 50 ug of OVA on 4 consecutive days (days 13, 14, 19, and 20). We mentioned "Immunized group" that were injected by I.P. with 100 ul of 10 ug/ml *Toxascaris leonina* excretory-secretory (Tl-ES) and *T. leonina* total protein (Tl-TP), respectively, on 7 and 14 days before airway allergic reaction procedure. Also "Challenged with OVA" group were mentioned which were injected by I.P. with 100 ul of 10 ug/ml of Tl-ES and Tl-TP respectively at the same challenge days in airway allergic reaction procedure. The negative control group was challenged with PBS (I.N.) on the same challenge day in airway allergic reaction procedure. A; PBS-treated, B-F; OVA+ alumtreated (induced asthma), (C) immunized Tl-ES protein, (D) immunized Tl-TP, (E) Tl-ES protein treatment with OVA challenge, (F) Tl-TP protein with OVA challenge.

IL-4 has been demonstrated to regulate isotype class switching in B cells to IgE synthesis, and IL-5 stimulates eosinophil growth, activates these cells, and prolongs eosinophil survival. *T. leonina-*derived proteins could inhibit increases in the levels of IL-4 and IL-5 from OVA challenge. In particular, the level of IL-4 in the BAL fluid in the immunized group was almost half of that observed in the OVA-only challenge group. This result was consistent with the results regarding the IgE concentration in the blood (15). Although levels of the IL-5 cytokine of *T. leonina* ES and total protein-immunized mice were lower than those observed in asthma

Parasite-Derived Proteins Inhibit Allergic Specific Th2 Response 535

Fig. 3. **Th1 and Th2 cytokines levels in the BAL fluids of Tl-ES and Tl-TP treated mice.** The levels of all Th2 cytokines of the immunized group were contrasted with those of the OVA-challenged control mice. The data were expressed as the means + SD of individual

How does helminth infection protect against allergy? Many hypotheses have been advanced thus far regarding this theme. One of these hypotheses is that non-specific IgE generated as the result of helminthic infection may inhibit allergen-specific IgE binding sites on mast cells or basophils. Although Jarrett suggested in 1980 that parasite-induced 'nonspecific' IgE does not protect against host allergic reactions (26). However, other scientists have suggested that nonspecific IgE can modulate host immune responses, as in the case of insulin-dependent diabetes, as well as Th2 response by allergens (25, 27-29). The other hypothesis states that helminth parasites stimulate the production of immunoregulatory mediators, which are likely to perform a function in the maintenance of the chronicity of infection, with no marked induction of pathology. In particular, elevated IL-10 levels have been associated previously with protection against allergic diseases in helminth-infected African children (4). Helminth infections can induce Treg cells from hosts, and these cells secrete IL-10 and suppress the proliferation of other CD4+ T cells (30-32). We also found that parasite proteins could also induce the IL-10 cytokine,

mice (\* ; p value <0.05, \*\*; p value <0.01).

particularly in the Tl-TP immunized group (Fig. 4).

control mice, this effect was not remarkable. Also, the total number of eosinophils was not substantially reduced as the result of immunization. Eosinophils and IgE proved vitally important in allergy-induced Th2 response; additionally, the elevation of the numbers of these cells and IgE levels were identified as specific responses to parasite infection and this response was shown to be elicited only by treatment with parasite total proteins (24, 25).

Fig. 2. **Airway hyperresponsiveness measurements and comparison of differential cell counts obtained via airway inflammation in ova-sensitized PBS mice and the effects in immunized and OVA-challenged groups**. Total protein of *T. leonina* immunization group [Tl-TP (I)] has lower penh value than those of OVA challenge group (A). The numbers of inflammatory cells were significantly lower in the parasite-derived protein-treated mice (B & C). The data were expressed as the means + SD of individual mice.

control mice, this effect was not remarkable. Also, the total number of eosinophils was not substantially reduced as the result of immunization. Eosinophils and IgE proved vitally important in allergy-induced Th2 response; additionally, the elevation of the numbers of these cells and IgE levels were identified as specific responses to parasite infection and this response

Fig. 2. **Airway hyperresponsiveness measurements and comparison of differential cell counts obtained via airway inflammation in ova-sensitized PBS mice and the effects in immunized and OVA-challenged groups**. Total protein of *T. leonina* immunization group [Tl-TP (I)] has lower penh value than those of OVA challenge group (A). The numbers of inflammatory cells were significantly lower in the parasite-derived protein-treated mice (B

& C). The data were expressed as the means + SD of individual mice.

was shown to be elicited only by treatment with parasite total proteins (24, 25).

Fig. 3. **Th1 and Th2 cytokines levels in the BAL fluids of Tl-ES and Tl-TP treated mice.** The levels of all Th2 cytokines of the immunized group were contrasted with those of the OVA-challenged control mice. The data were expressed as the means + SD of individual mice (\* ; p value <0.05, \*\*; p value <0.01).

How does helminth infection protect against allergy? Many hypotheses have been advanced thus far regarding this theme. One of these hypotheses is that non-specific IgE generated as the result of helminthic infection may inhibit allergen-specific IgE binding sites on mast cells or basophils. Although Jarrett suggested in 1980 that parasite-induced 'nonspecific' IgE does not protect against host allergic reactions (26). However, other scientists have suggested that nonspecific IgE can modulate host immune responses, as in the case of insulin-dependent diabetes, as well as Th2 response by allergens (25, 27-29). The other hypothesis states that helminth parasites stimulate the production of immunoregulatory mediators, which are likely to perform a function in the maintenance of the chronicity of infection, with no marked induction of pathology. In particular, elevated IL-10 levels have been associated previously with protection against allergic diseases in helminth-infected African children (4). Helminth infections can induce Treg cells from hosts, and these cells secrete IL-10 and suppress the proliferation of other CD4+ T cells (30-32). We also found that parasite proteins could also induce the IL-10 cytokine, particularly in the Tl-TP immunized group (Fig. 4).

Parasite-Derived Proteins Inhibit Allergic Specific Th2 Response 537

MIF was described initially as one of the earliest cytokines to be derived from activated T-cells, and was believed to prevent the random migration of macrophages (42). MIF has also been demonstrated to be generated abundantly by monocytes/macrophages and to function in an autocrine/paracrine manner in the upregulation and sustenance of the activation of diverse cell types (43). The profile of the activities of MIF, both *in vivo* and *in vitro,* is reflective of a role for MIF in the pathogenesis of a variety of inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, ankylosing spondylitis, and psoriasis (44). MIF performs a crucial function in airway inflammation and airway hyper-responsiveness in asthma (45). Recently, several MIF homologues have been isolated from parasitic nematodes. Two types of MIF homologues have thus far been identified in nematodes (34). The type 1 MIF homologues bear a greater amino acid similarity with the mammalian MIFs than do the type 2 MIF homologues (46). The type 1 MIF homologues isolated from *Brugia malayi* (Bm-MIF1) induce eosinophil recruitment, and alternatively activated macrophage recruitment *in vivo*  when injected into the peritoneal cavities of mice. Mutation of the conserved proline residue induces the abrogation of this activity (47). This ability of parasite MIF homologues was similar to those of mammalian MIF. However, recently Cho et al. reported that hookworm

MIF (structurally type 2 MIF) functions differently from mammalian MIF (48, 49).

to the function of host MIF (59).

treatment in OVA-alum asthma-induced mice (Fig. 6).

We have cloned another type 2 MIF homologue (As-MIF) from *Anisakis simplex* (whale worm) 3rd stage larva, which causes anisakidosis in humans (50). Recombinant As-MIF (rAs-MIF) proved highly effective with regard to the inhibition of goblet cell hyperplasia and inflammatory responses in the airways of OVA-induced asthma model mice (Fig. 5). Increasing concentrations of rAs-MIF induced an increase in the anti-inflammatory effects on asthma model mice. Additionally, the function of rAs-MIF was antagonized as compared

How does As-MIF suppress allergy responses in mice? There have been many reports demonstrating that Th2-type effector responses may be regulated by Treg cells (51-53). Additionally, nematode infections can induce and expand naturally occurring Treg cells in both humans and mice (4, 54), thereby suggesting a role for these Treg cells in the helminthinduced modulation of inflammatory diseases (55, 56). In particular, the clinical symptoms of allergic airway inflammation in the mouse model was clearly modulated by Treg cell mediated immune suppression, which was itself activated by helminth infection or antigen treatment (57, 58). We could determine the increase in Treg cells as the result of rAs-MIF

IL-10 and TGF-β were produced primarily by Treg cells, and they are known to suppress immune response effects. The IL-10 and TGF-β levels measured in BALFs from rAs-MIFtreated asthma-induced mice were higher than those of the asthma-induced mice; the IL-10 and TGF-β levels occurred in accordance with their treated concentrations (59). The helminthic parasites stimulate the production of immunoregulatory mediators, which likely perform a function in the maintenance of the chronicity of infection, without any marked induction of pathology. In particular, elevated IL-10 levels have been associated with responses against allergic diseases in helminth-infected individuals (4). Also, Nagler-Anderson *et al*. showed that in mice sensitized with peanut plus cholera toxin, anti-IL-10 treatment abrogated the ability of helminths to protect against allergic symptoms and to downregulate allergen-specific IgE. IL-10, which is referred to as the cytokine synthesis inhibitory factor, is an anti-inflammatory cytokine, which is capable of inhibiting the

Fig. 4. **Treg cell related cytokines levels in the BAL fluids of Tl-ES and Tl-TP treated mice.**  The data were expressed as the means + SD of individual mice (\* ; p value <0.05, \*\*; p value <0.01).

Finally, the data presented herein demonstrate that *T. leonina*-derived proteins may perform a crucial function in resistance against Th2 immune responses. We suggested that this inhibition may be related to the IL-10 cytokine, which was induced by parasite proteins. Further steps are currently being taken in an effort to gain a greater understanding of the molecular basis of immune evasion by nematodes. Thus, we are attempting to gain new insights into the immune regulation strategies of nematodes, and the growing number of new strategies employed by parasites to exert their marked downregulatory effects.

#### **3. Macrophage migration inhibitory factor homologues of parasite suppress Th2 response in allergic airway inflammation model via Treg cell recruitment**

A number of parasite-derived proteins, glycoconjugates, and small lipid moieties have been demonstrated to perform known or hypothesized functions in immune interference. Other researchers have already isolated several other immune downregulatory molecules from parasites, and these molecules have been identified as mammal cytokine homologues, protease inhibitors, abundant larval transcript antigens, glyco-networks, and venom allergen-like proteins (33-39). The cytokine network is a crucial component of host defense against pathogens. It is not, therefore, surprising to find that one of the immune evasion strategies utilized by infectious organisms is the generation of mammalian cytokine homologues, including TGF-β and the macrophage migration inhibitory factor (MIF) (40, 41).

Fig. 4. **Treg cell related cytokines levels in the BAL fluids of Tl-ES and Tl-TP treated mice.**  The data were expressed as the means + SD of individual mice (\* ; p value <0.05, \*\*; p value

Finally, the data presented herein demonstrate that *T. leonina*-derived proteins may perform a crucial function in resistance against Th2 immune responses. We suggested that this inhibition may be related to the IL-10 cytokine, which was induced by parasite proteins. Further steps are currently being taken in an effort to gain a greater understanding of the molecular basis of immune evasion by nematodes. Thus, we are attempting to gain new insights into the immune regulation strategies of nematodes, and the growing number of new strategies employed by parasites to exert their marked down-

**3. Macrophage migration inhibitory factor homologues of parasite suppress Th2 response in allergic airway inflammation model via Treg cell recruitment**  A number of parasite-derived proteins, glycoconjugates, and small lipid moieties have been demonstrated to perform known or hypothesized functions in immune interference. Other researchers have already isolated several other immune downregulatory molecules from parasites, and these molecules have been identified as mammal cytokine homologues, protease inhibitors, abundant larval transcript antigens, glyco-networks, and venom allergen-like proteins (33-39). The cytokine network is a crucial component of host defense against pathogens. It is not, therefore, surprising to find that one of the immune evasion strategies utilized by infectious organisms is the generation of mammalian cytokine homologues, including TGF-β and the macrophage migration inhibitory factor (MIF)

<0.01).

regulatory effects.

(40, 41).

MIF was described initially as one of the earliest cytokines to be derived from activated T-cells, and was believed to prevent the random migration of macrophages (42). MIF has also been demonstrated to be generated abundantly by monocytes/macrophages and to function in an autocrine/paracrine manner in the upregulation and sustenance of the activation of diverse cell types (43). The profile of the activities of MIF, both *in vivo* and *in vitro,* is reflective of a role for MIF in the pathogenesis of a variety of inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, ankylosing spondylitis, and psoriasis (44). MIF performs a crucial function in airway inflammation and airway hyper-responsiveness in asthma (45).

Recently, several MIF homologues have been isolated from parasitic nematodes. Two types of MIF homologues have thus far been identified in nematodes (34). The type 1 MIF homologues bear a greater amino acid similarity with the mammalian MIFs than do the type 2 MIF homologues (46). The type 1 MIF homologues isolated from *Brugia malayi* (Bm-MIF1) induce eosinophil recruitment, and alternatively activated macrophage recruitment *in vivo*  when injected into the peritoneal cavities of mice. Mutation of the conserved proline residue induces the abrogation of this activity (47). This ability of parasite MIF homologues was similar to those of mammalian MIF. However, recently Cho et al. reported that hookworm MIF (structurally type 2 MIF) functions differently from mammalian MIF (48, 49).

We have cloned another type 2 MIF homologue (As-MIF) from *Anisakis simplex* (whale worm) 3rd stage larva, which causes anisakidosis in humans (50). Recombinant As-MIF (rAs-MIF) proved highly effective with regard to the inhibition of goblet cell hyperplasia and inflammatory responses in the airways of OVA-induced asthma model mice (Fig. 5). Increasing concentrations of rAs-MIF induced an increase in the anti-inflammatory effects on asthma model mice. Additionally, the function of rAs-MIF was antagonized as compared to the function of host MIF (59).

How does As-MIF suppress allergy responses in mice? There have been many reports demonstrating that Th2-type effector responses may be regulated by Treg cells (51-53). Additionally, nematode infections can induce and expand naturally occurring Treg cells in both humans and mice (4, 54), thereby suggesting a role for these Treg cells in the helminthinduced modulation of inflammatory diseases (55, 56). In particular, the clinical symptoms of allergic airway inflammation in the mouse model was clearly modulated by Treg cell mediated immune suppression, which was itself activated by helminth infection or antigen treatment (57, 58). We could determine the increase in Treg cells as the result of rAs-MIF treatment in OVA-alum asthma-induced mice (Fig. 6).

IL-10 and TGF-β were produced primarily by Treg cells, and they are known to suppress immune response effects. The IL-10 and TGF-β levels measured in BALFs from rAs-MIFtreated asthma-induced mice were higher than those of the asthma-induced mice; the IL-10 and TGF-β levels occurred in accordance with their treated concentrations (59). The helminthic parasites stimulate the production of immunoregulatory mediators, which likely perform a function in the maintenance of the chronicity of infection, without any marked induction of pathology. In particular, elevated IL-10 levels have been associated with responses against allergic diseases in helminth-infected individuals (4). Also, Nagler-Anderson *et al*. showed that in mice sensitized with peanut plus cholera toxin, anti-IL-10 treatment abrogated the ability of helminths to protect against allergic symptoms and to downregulate allergen-specific IgE. IL-10, which is referred to as the cytokine synthesis inhibitory factor, is an anti-inflammatory cytokine, which is capable of inhibiting the

Parasite-Derived Proteins Inhibit Allergic Specific Th2 Response 539

Fig. 6. Treg cell production could be induced by rAs-MIF treatment. Treg cell populations in the lungs and spleen were significantly increased by rAs-MIF treatment, but this effect was inhibited by rAnti-As-MIF. (\*; p < 0. 05, \*\*; p < 0.01, n = 5 mice per group, 3 independent

We showed that parasite derived proteins may perform a crucial function in resistance against allergic airway inflammation via IL-10 cytokine induction and Treg cell recruitment. Parasites regulate or suppress their host immune response, maintaining their parasitism for a prolonged period, using unknown molecules. As-MIF might be one of the molecules that affect host immune regulation. The further characterization of parasite derived proteins might ultimately result in the design of novel therapeutic intervention strategies for the

[1] Zaccone, P., Z. Fehervari, J. M. Phillips, D. W. Dunne, and A. Cooke. 2006. Parasitic

[2] Scrivener, S., H. Yemaneberhan, M. Zebenigus, D. Tilahun, S. Girma, S. Ali, P.

[3] Dagoye, D., Z. Bekele, K. Woldemichael, H. Nida, M. Yimam, A. Hall, A. J. Venn, J. R.

McElroy, A. Custovic, A. Woodcock, D. Pritchard, A. Venn, and J. Britton. 2001. Independent effects of intestinal parasite infection and domestic allergen exposure on risk of wheeze in Ethiopia: a nested case-control study. *Lancet*

Britton, R. Hubbard, and S. A. Lewis. 2003. Wheezing, allergy, and parasite

worms and inflammatory diseases. *Parasite immunology* 28:515-523.

experiments).

**4. Conclusion**

treatment of asthma.

358:1493-1499.

**5. References**

synthesis of pro-inflammatory cytokines (60). The IL-10 requirement is critical to several important human diseases, including schistosomiasis, wherein marked increases in host morbidity and mortality are observed when IL-10 levels are low or absent (61). In cases of murine *S. mansoni* infection, IL-10 attenuates the hepatocyte damage induced by the eggs of the parasite. IL-10 is also essential for the maintenance of non-lethal chronic infections, in addition to the inhibition of inappropriate immune responses in experimental models (62). TGF-β1 is also a strong candidate for immune suppression by Treg cells from helminthinfected mice, and has already been recognized to alleviate experimental airway allergy symptoms (63) and to instruct peripheral T cells to develop their regulatory capacities (64). Thus, the inhibition of asthma response by rAs-MIF may be associated with the principal Treg cell–associated downregulatory cytokines, including TGF-β1 and IL-10.

Fig. 5. **Histologic appearance of lungs after challenge with PBS, OVA, and rAs-MIF by concentration (H-E stain).** (A; x 100, B; x 600; C; PAS stain), C; **a**, **f**, and **k**; phosphatebuffered saline (PBS) treated, **b-e**, **g-j**, and **l-o**; OVA plus alum-treated (induced asthma), **c**, **h** and **m**; challenged with 1 μg/ml rAs-MIF, **d**, **i**, and **n**; challenged with 10 μg/ml rAs-MIF. **e**, **j**, and **o**; challenged with 100μg/ml rAs-MIF. In asthma-induced mice, a massive peribronchial infiltration with immune-related cells and hyperplasia of bronchial epithelial cells were observed. Upon challenge with 1μg/ml rAs-MIF treatment (**c**, **h** and **m**), asthmainduced mice evidenced thinner bronchial epithelial cells than were observed in the asthmainduced mice (**b**, **g**, and **l**). Mice challenged with treatment with 10 and 100μg/ml rAs-MIF evidenced thinner than normal bronchial epithelial cells and decreased numbers of immunerelated cells. Goblet cells and immune-related cells in the airway walls of mice exposed to PBS, OVA and OVA challenge with 1, 10, and 100 μg/ml rAs-MIF. In asthma-induced mice (g), a massive peri-bronchial infiltration of inflammatory cells and hyperplasia of bronchial epithelial cells were detected. However, goblet cell hyperplasia was reduced in the bronchial epithelial cells of the rAs-MIF-treated mice (h-j).

Fig. 6. Treg cell production could be induced by rAs-MIF treatment. Treg cell populations in the lungs and spleen were significantly increased by rAs-MIF treatment, but this effect was inhibited by rAnti-As-MIF. (\*; p < 0. 05, \*\*; p < 0.01, n = 5 mice per group, 3 independent experiments).

#### **4. Conclusion**

538 Allergic Diseases – Highlights in the Clinic, Mechanisms and Treatment

synthesis of pro-inflammatory cytokines (60). The IL-10 requirement is critical to several important human diseases, including schistosomiasis, wherein marked increases in host morbidity and mortality are observed when IL-10 levels are low or absent (61). In cases of murine *S. mansoni* infection, IL-10 attenuates the hepatocyte damage induced by the eggs of the parasite. IL-10 is also essential for the maintenance of non-lethal chronic infections, in addition to the inhibition of inappropriate immune responses in experimental models (62). TGF-β1 is also a strong candidate for immune suppression by Treg cells from helminthinfected mice, and has already been recognized to alleviate experimental airway allergy symptoms (63) and to instruct peripheral T cells to develop their regulatory capacities (64). Thus, the inhibition of asthma response by rAs-MIF may be associated with the principal

Treg cell–associated downregulatory cytokines, including TGF-β1 and IL-10.

Fig. 5. **Histologic appearance of lungs after challenge with PBS, OVA, and rAs-MIF by concentration (H-E stain).** (A; x 100, B; x 600; C; PAS stain), C; **a**, **f**, and **k**; phosphate-

epithelial cells of the rAs-MIF-treated mice (h-j).

buffered saline (PBS) treated, **b-e**, **g-j**, and **l-o**; OVA plus alum-treated (induced asthma), **c**, **h** and **m**; challenged with 1 μg/ml rAs-MIF, **d**, **i**, and **n**; challenged with 10 μg/ml rAs-MIF. **e**, **j**, and **o**; challenged with 100μg/ml rAs-MIF. In asthma-induced mice, a massive peribronchial infiltration with immune-related cells and hyperplasia of bronchial epithelial cells were observed. Upon challenge with 1μg/ml rAs-MIF treatment (**c**, **h** and **m**), asthmainduced mice evidenced thinner bronchial epithelial cells than were observed in the asthmainduced mice (**b**, **g**, and **l**). Mice challenged with treatment with 10 and 100μg/ml rAs-MIF evidenced thinner than normal bronchial epithelial cells and decreased numbers of immunerelated cells. Goblet cells and immune-related cells in the airway walls of mice exposed to PBS, OVA and OVA challenge with 1, 10, and 100 μg/ml rAs-MIF. In asthma-induced mice (g), a massive peri-bronchial infiltration of inflammatory cells and hyperplasia of bronchial epithelial cells were detected. However, goblet cell hyperplasia was reduced in the bronchial We showed that parasite derived proteins may perform a crucial function in resistance against allergic airway inflammation via IL-10 cytokine induction and Treg cell recruitment. Parasites regulate or suppress their host immune response, maintaining their parasitism for a prolonged period, using unknown molecules. As-MIF might be one of the molecules that affect host immune regulation. The further characterization of parasite derived proteins might ultimately result in the design of novel therapeutic intervention strategies for the treatment of asthma.

#### **5. References**

Parasite-Derived Proteins Inhibit Allergic Specific Th2 Response 541

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[19] Wang, C. C., T. J. Nolan, G. A. Schad, and D. Abraham. 2001. Infection of mice with the

[20] Kobayashi, Y., S. Ishizaki, K. Shimakura, Y. Nagashima, and K. Shiomi. 2007. Molecular

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[22] Furmonaviciene, R., H. F. Sewell, and F. Shakib. 2000. Comparative molecular

[23] Holt, D. C., K. Fischer, G. E. Allen, D. Wilson, P. Wilson, R. Slade, B. J. Currie, S. F.

[24] Ishizaka, T., J. Urban, Jr., K. Takatsu, and K. Ishizaka. 1976. Immunoglobulin E

[25] Ehigiator, H. N., A. W. Stadnyk, and T. D. Lee. 2000. Extract of Nippostrongylus

[26] Jarrett, E., S. Mackenzie, and H. Bennich. 1980. Parasite-induced 'nonspecific' IgE does

[27] Imai, S., H. Tezuka, Y. Furuhashi, R. Muto, and K. Fujita. 2001. A factor of inducing IgE

[28] Imai, S., H. Tezuka, and K. Fujita. 2001. A factor of inducing IgE from a filarial parasite

colubriformis. *International journal for parasitology* 33:1233-1243.

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**27**

**Pharmaceutical Treatment of Asthma**

Jimmi Elers and Vibeke Backer

*Copenhagen University,*

*Denmark*

**Symptoms in Elite Athletes – Doping or Therapy**

According to the World Health Organization (WHO), 300 million people suffer from asthma, a disease which is increasing in western societies, and furthermore asthma is the most common chronic disease among children, adolescents and young adults. A steady increase in the prevalence of asthma, has been seen in most countries in recent decades (Thomsen et al 2011) and the higher frequency of asthma in young people may partly explain the high frequency found in elite athletes – although both frequency of asthma symptoms and use of antiasthmatic medication are different than expected. Moreover, the frequency of airway hyperresponsiveness (AHR) in elite athletes is higher than expected as well as the frequency of asthma-like symptoms (cough, wheeze, shortness of breath and chest tightness) which might

The frequency of asthma among the general population is around 7-10%; whereas the frequency of asthma among elite athletes is found to be higher, especially among endurance athletes (Pedersen et al 2008b). It seems as asthma is something they gain, as only one third of Olympic athletes had childhood asthma. Although asthma among the general public is a permanent phenomenon, it seems to be different in elite athletes, as asthma apparently

Asthma is a chronic respiratory condition classically characterised by airway inflammation and airway hyperresponsiveness (AHR) to multiple stimuli (Anderson et al 2009). AHR is defined as a pathological bronchoconstrictive response to a given stimulus . AHR to a direct stimulus (e.g. methacholine) acts through airway smooth muscles and the response is thought to be independent of airway inflammation. Whereas response to an indirect stimulus (e.g. exercise, hyperventilation, mannitol) acts through a release of inflammatory mediators, such as histamine, prostaglandins, and leukotrienes, which causes contraction of the airway smooth muscle cells. Airway inflammation in asthma is characterised by inflammatory cells, such as eosinophils, mast cells, and macrophages. The inflammation causes remodelling of the basal membrane, enlargement of the mass of smooth muscles, and disturbance of the surface area. Permanent use of controller therapy with inhaled steroids (ICS) is needed in asthma, as well as relief therapy with short-acting beta2-agonists (SABA),

be caused by exhaustive ventilation, but the pathogenesis is still unknown.

disappeared after retiring from the sport (Fitch et al 2008).

**2. Asthma among athletes**

long-acting beta2-agonists (LABA) or others.

**1. Introduction**

### **Pharmaceutical Treatment of Asthma Symptoms in Elite Athletes – Doping or Therapy**

Jimmi Elers and Vibeke Backer *Copenhagen University, Denmark*

#### **1. Introduction**

544 Allergic Diseases – Highlights in the Clinic, Mechanisms and Treatment

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2006. Intestinal helminths protect in a murine model of asthma. *J Immunol*

Jeong, M. H. Lee, and H. S. Yu. 2009. Macrophage migration inhibitory factor homologs of anisakis simplex suppress Th2 response in allergic airway inflammation model via CD4+CD25+Foxp3+ T cell recruitment. *J Immunol*

producing IL-10 mediate tolerance induced by respiratory exposure to antigen.

Golovkina, C. A. Hunter, and A. Thomas-Tikhonenko. 2003. An essential role of Th1 responses and interferon gamma in infection-mediated suppression of

DeKruyff, and D. T. Umetsu. 2000. CD4(+) T helper cells engineered to produce latent TGF-beta1 reverse allergen-induced airway hyperreactivity and

Wahl. 2003. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. *The Journal*  According to the World Health Organization (WHO), 300 million people suffer from asthma, a disease which is increasing in western societies, and furthermore asthma is the most common chronic disease among children, adolescents and young adults. A steady increase in the prevalence of asthma, has been seen in most countries in recent decades (Thomsen et al 2011) and the higher frequency of asthma in young people may partly explain the high frequency found in elite athletes – although both frequency of asthma symptoms and use of antiasthmatic medication are different than expected. Moreover, the frequency of airway hyperresponsiveness (AHR) in elite athletes is higher than expected as well as the frequency of asthma-like symptoms (cough, wheeze, shortness of breath and chest tightness) which might be caused by exhaustive ventilation, but the pathogenesis is still unknown.

The frequency of asthma among the general population is around 7-10%; whereas the frequency of asthma among elite athletes is found to be higher, especially among endurance athletes (Pedersen et al 2008b). It seems as asthma is something they gain, as only one third of Olympic athletes had childhood asthma. Although asthma among the general public is a permanent phenomenon, it seems to be different in elite athletes, as asthma apparently disappeared after retiring from the sport (Fitch et al 2008).

#### **2. Asthma among athletes**

Asthma is a chronic respiratory condition classically characterised by airway inflammation and airway hyperresponsiveness (AHR) to multiple stimuli (Anderson et al 2009). AHR is defined as a pathological bronchoconstrictive response to a given stimulus . AHR to a direct stimulus (e.g. methacholine) acts through airway smooth muscles and the response is thought to be independent of airway inflammation. Whereas response to an indirect stimulus (e.g. exercise, hyperventilation, mannitol) acts through a release of inflammatory mediators, such as histamine, prostaglandins, and leukotrienes, which causes contraction of the airway smooth muscle cells. Airway inflammation in asthma is characterised by inflammatory cells, such as eosinophils, mast cells, and macrophages. The inflammation causes remodelling of the basal membrane, enlargement of the mass of smooth muscles, and disturbance of the surface area. Permanent use of controller therapy with inhaled steroids (ICS) is needed in asthma, as well as relief therapy with short-acting beta2-agonists (SABA), long-acting beta2-agonists (LABA) or others.

Pharmaceutical Treatment of Asthma Symptoms in Elite Athletes – Doping or Therapy 547

sometimes in subjects suffering from an extrathoracal disorder such as vocal cord

In conclusion, the most frequent complaint among healthy and diseased athletes is respiratory symptoms during exercise. Furthermore, healthy elite athletes often have AHR with a pattern which differ from the general asthma patient. Lastly, the types of cells

Not all that wheezes is asthma. When the diagnosis of asthma is based on respiratory symptoms alone, misdiagnosis may occur. Patients may present with respiratory distress, such as wheezy, when experiencing a low level of fitness, a psychological condition, inhalation of airborne irritants, rhinosinusitis, or gastroesophageal reflux disease. Moreover, exercise-induced symptoms can occur as periodic occurance of laryngeal obstruction (POLO) or exercise induced laryngeal obstruction (EILO) and present with asthma like symptoms. These diseases include conditions such as vocal cord dysfunction (VCD), exercise-induced paradoxical arytenoid motion (EPAM), exercise-induced laryngomalacia (EIL), exercise-induced laryngochalasia, angioedema, vocal cord tumours, and vocal cord paralysis. There seems to be a substantial overlap between EILO and EIA, at least in elite athletes. It could be of minor importance, but it could also have a major influence on the diagnostic procedure in the daily care settings of those with asthma-like symptoms. When POLO/EILO is misdiagnosed as asthma, patient are erroneously treated with anti-asthma

These diseases are easily recognized by performing a Flow/Volume curve where a classical cutoff of the inspiratory loop is apparent. On the other hand, a specific diagnose of the actual pathology need laryngeal examination during exercise. The definitive diagnosis of laryngeal obstruction might require laryngoscopy during strenuous exercise. Heimdal et al (Heimdal et al 2006) recently published a paper describing a model for use when performing the continuous laryngoscopy exercise test (CLE). Patients with asthma should start asthma medication, and for those with satisfactory adherence who do not achieve wellcontrolled disease, other reasons for persistent respiratory symptoms should be explored. In

Treatment of elite athletes with asthma can be divided into non-pharmacological and pharmacological treatment. During the last decades the International Olympic Committee (IOC) increased their focus on the increasing use of anti-asthmatic medication by Olympic and other elite athletes. At the 1996 Olympic Games in Atlanta 3.7% of the athletes used beta2-agonists, 5.6% at the 1998 Winter Games in Nagano, and 5.7% at the Sydney Games 2000. The IOC and the World Anti Doping Agency (WADA) have had many changes in the anti-doping regulations on beta2-agonists through the years, partly due to concerns regarding ergogenic effects and partly because of health risk concerns. Due to the increased use of beta2-agonists as mentioned above the IOC introduced a criterion of demonstration of asthma by an objective measure of reversibility or bronchial airway hyperresponsiveness in order approved use of beta2-agonist. This resulted in a 27% reduction in the use of beta2-

involved in the inflammation are different from those in normal asthmatics.

**4. Diseases mimicking exercise-induced asthma**

therapy, even high doses because of "resistant disease".

such cases CLE should be performed.

agonists in the 2004 Games in Athen.

**5. Treatment**

dysfunction (VCD).

Elite athletes, with or without asthma, have asthma-like symptoms during their training season, especially cough, phlegm and shortness of breath is a dominant complain among athletes (Lund et al 2009). Furthermore, athletes are more often found with AHR to direct stimuli and less to direct stimuli than found in normal subjects thus suggesting a respiratory illness. Variation in action of the different provocative agents is related to the fact that response to an indirect challenge would reflect an ongoing inflammation better than would direct tests, and perhaps thus reflect the presence of classical asthma, whereas AHR to direct agents indicates airway smooth muscle dysfunction and, in some cases, asthma (Pedersen et al 2008a, Sue-Chu et al 2010).

The definition of asthma includes respiratory symptoms, variable airway obstruction and airway hyperresponsiveness to multiple agents. The respiratory symptoms in patients with common asthma and the elite athletes with or without asthma are similar, which leave a diagnostic problem in these cases where symptoms are the single diagnostic parameter available. Whereas, the differences between common asthma and asthma among elite athletes are predominantly related to the airway responsiveness to inhaled agents, the dayto-day variability of lung function as well as the content of the inflammatory cells which predominantly are neutrophilic cells and not eosinophilic.

#### **3. Exercise-induced asthma and bronchoconstriction in elite athletes**

Exercise-induced bronchoconstriction (EIB) is an acute, transient narrowing of the airway that occurs during and particularly after exercise. In most scientific papers, exercise-induced asthma (EIA) are defined as respiratory symptoms and a significant reduction in FEV1 after exercise, i.e. ∆ FEV1 ≥ 10%, whereas EIB is a significant reduction in FEV1 (≥10%) when tested, independent of symptoms or not. In population samples, the prevalence of EIB (16%) is the same as the findings of AHR to histamine (16%), although only 6% had responsiveness to both test (Backer et al 1992). EIB is believed to be more specific to asthma, but less sensitive, as the number of false negative results is a problem when research and clinical situations settings are evaluated (Anderson & Kippelen 2005). The frequency of AHR is frequently found above 40%, which is higher than the frequency of asthma. Elite athletes claim that exercise is the most prominent trigger of asthma symptoms, as they very seldom complain of respiratory symptoms at rest or during the night.

The pathogenesis of asthma-like symptoms in elite athletes is multifactorial, and is not completely understood. However, deep, exhaustive ventilation during exercise brings atmospheric air which is cold and dry and this manoeuvre overcomes the ability of the upper airways to warm up and humidify the air reaching the smaller airways(Anderson & Kippelen 2005). This brings about airway narrowing due to osmotic and thermal evaporative water loss, and some vascular involvement. These airway differences, together with some degree of inflammation, lead to a respiratory condition described as EIB or sports asthma. This abnormal response is most often found in endurance sports, such as cross-country skiing , swimming, rowing, cycling, fast-track skating and long distance running.

The symptoms of exercise-induced bronchoconstriction range from mild impairment of performance with minor reduction in lung function after exercise to severe bronchospasm with large reduction in FEV1. In athletes, however, the most common symptoms include cough, wheezing, chest tightness, dyspnoea, and fatigue. These symptoms are frequently found in healthy subjects, subjects with asthma, subjects who are not in good condition and sometimes in subjects suffering from an extrathoracal disorder such as vocal cord dysfunction (VCD).

In conclusion, the most frequent complaint among healthy and diseased athletes is respiratory symptoms during exercise. Furthermore, healthy elite athletes often have AHR with a pattern which differ from the general asthma patient. Lastly, the types of cells involved in the inflammation are different from those in normal asthmatics.

#### **4. Diseases mimicking exercise-induced asthma**

Not all that wheezes is asthma. When the diagnosis of asthma is based on respiratory symptoms alone, misdiagnosis may occur. Patients may present with respiratory distress, such as wheezy, when experiencing a low level of fitness, a psychological condition, inhalation of airborne irritants, rhinosinusitis, or gastroesophageal reflux disease. Moreover, exercise-induced symptoms can occur as periodic occurance of laryngeal obstruction (POLO) or exercise induced laryngeal obstruction (EILO) and present with asthma like symptoms. These diseases include conditions such as vocal cord dysfunction (VCD), exercise-induced paradoxical arytenoid motion (EPAM), exercise-induced laryngomalacia (EIL), exercise-induced laryngochalasia, angioedema, vocal cord tumours, and vocal cord paralysis. There seems to be a substantial overlap between EILO and EIA, at least in elite athletes. It could be of minor importance, but it could also have a major influence on the diagnostic procedure in the daily care settings of those with asthma-like symptoms. When POLO/EILO is misdiagnosed as asthma, patient are erroneously treated with anti-asthma therapy, even high doses because of "resistant disease".

These diseases are easily recognized by performing a Flow/Volume curve where a classical cutoff of the inspiratory loop is apparent. On the other hand, a specific diagnose of the actual pathology need laryngeal examination during exercise. The definitive diagnosis of laryngeal obstruction might require laryngoscopy during strenuous exercise. Heimdal et al (Heimdal et al 2006) recently published a paper describing a model for use when performing the continuous laryngoscopy exercise test (CLE). Patients with asthma should start asthma medication, and for those with satisfactory adherence who do not achieve wellcontrolled disease, other reasons for persistent respiratory symptoms should be explored. In such cases CLE should be performed.

#### **5. Treatment**

546 Allergic Diseases – Highlights in the Clinic, Mechanisms and Treatment

Elite athletes, with or without asthma, have asthma-like symptoms during their training season, especially cough, phlegm and shortness of breath is a dominant complain among athletes (Lund et al 2009). Furthermore, athletes are more often found with AHR to direct stimuli and less to direct stimuli than found in normal subjects thus suggesting a respiratory illness. Variation in action of the different provocative agents is related to the fact that response to an indirect challenge would reflect an ongoing inflammation better than would direct tests, and perhaps thus reflect the presence of classical asthma, whereas AHR to direct agents indicates airway smooth muscle dysfunction and, in some cases, asthma (Pedersen et

The definition of asthma includes respiratory symptoms, variable airway obstruction and airway hyperresponsiveness to multiple agents. The respiratory symptoms in patients with common asthma and the elite athletes with or without asthma are similar, which leave a diagnostic problem in these cases where symptoms are the single diagnostic parameter available. Whereas, the differences between common asthma and asthma among elite athletes are predominantly related to the airway responsiveness to inhaled agents, the dayto-day variability of lung function as well as the content of the inflammatory cells which

**3. Exercise-induced asthma and bronchoconstriction in elite athletes**

Exercise-induced bronchoconstriction (EIB) is an acute, transient narrowing of the airway that occurs during and particularly after exercise. In most scientific papers, exercise-induced asthma (EIA) are defined as respiratory symptoms and a significant reduction in FEV1 after exercise, i.e. ∆ FEV1 ≥ 10%, whereas EIB is a significant reduction in FEV1 (≥10%) when tested, independent of symptoms or not. In population samples, the prevalence of EIB (16%) is the same as the findings of AHR to histamine (16%), although only 6% had responsiveness to both test (Backer et al 1992). EIB is believed to be more specific to asthma, but less sensitive, as the number of false negative results is a problem when research and clinical situations settings are evaluated (Anderson & Kippelen 2005). The frequency of AHR is frequently found above 40%, which is higher than the frequency of asthma. Elite athletes claim that exercise is the most prominent trigger of asthma symptoms, as they very seldom

The pathogenesis of asthma-like symptoms in elite athletes is multifactorial, and is not completely understood. However, deep, exhaustive ventilation during exercise brings atmospheric air which is cold and dry and this manoeuvre overcomes the ability of the upper airways to warm up and humidify the air reaching the smaller airways(Anderson & Kippelen 2005). This brings about airway narrowing due to osmotic and thermal evaporative water loss, and some vascular involvement. These airway differences, together with some degree of inflammation, lead to a respiratory condition described as EIB or sports asthma. This abnormal response is most often found in endurance sports, such as cross-country skiing , swimming, rowing, cycling, fast-track skating and long

The symptoms of exercise-induced bronchoconstriction range from mild impairment of performance with minor reduction in lung function after exercise to severe bronchospasm with large reduction in FEV1. In athletes, however, the most common symptoms include cough, wheezing, chest tightness, dyspnoea, and fatigue. These symptoms are frequently found in healthy subjects, subjects with asthma, subjects who are not in good condition and

al 2008a, Sue-Chu et al 2010).

distance running.

predominantly are neutrophilic cells and not eosinophilic.

complain of respiratory symptoms at rest or during the night.

Treatment of elite athletes with asthma can be divided into non-pharmacological and pharmacological treatment. During the last decades the International Olympic Committee (IOC) increased their focus on the increasing use of anti-asthmatic medication by Olympic and other elite athletes. At the 1996 Olympic Games in Atlanta 3.7% of the athletes used beta2-agonists, 5.6% at the 1998 Winter Games in Nagano, and 5.7% at the Sydney Games 2000. The IOC and the World Anti Doping Agency (WADA) have had many changes in the anti-doping regulations on beta2-agonists through the years, partly due to concerns regarding ergogenic effects and partly because of health risk concerns. Due to the increased use of beta2-agonists as mentioned above the IOC introduced a criterion of demonstration of asthma by an objective measure of reversibility or bronchial airway hyperresponsiveness in order approved use of beta2-agonist. This resulted in a 27% reduction in the use of beta2 agonists in the 2004 Games in Athen.

Pharmaceutical Treatment of Asthma Symptoms in Elite Athletes – Doping or Therapy 549

Inhaled long acting beta2-agonists (LABA), e.g. salmeterol and formoterol, are used in management of uncontrolled asthma treated with inhaled corticosteroid alone. LABA is used as add-on therapy to inhaled corticosteroids, either as fixed combination or in two separate devices. LABA should never be used as monotherapy in asthma due to risk of serious adverse events with increased risk of mortality in case of exacerbation. In a review from 2005 it was concluded that combined fluticasone and salmeterol was superior to fluticasone as monotherapy in preventing EIA. Furthermore, in a randomized, doubleblinded study combined budesonide/formoterol was compared with budesonide alone, asthma control was better with reduced symptoms when treated with combination of budesonide/formoterol. A new study from 2010 confirms previous findings with more efficacy when inhaled budesonide is combined with formoterol among adults and adolescents with moderate to severe asthma. These findings indicate that it is not an effect

related to the specific drug, but a class effect of the combination.

**(IN- AND OUT-OF-COMPETITION):**

S4. Hormone antagonists and modulators S5. Diuretics and other masking agents M1. Enhancement of oxygen transfer M2. Chemical and physical manipulation

S0. Non-approved substances

S0-5 and M1-3 defined above

S9. Glucocorticosterooids

S1. Anabolic agents

S3. Beta2-agonists

M3. Gene doping

S6. Stimulants S7. Narcotics S8. Cannabinoids

P1. Alcohol P2. Beta-Blockers

**I SUBSTANCES AND METHODS PROHIBITED AT ALL TIMES**

**II SUBSTANCES AND METHODS PROHIBITED IN-COMPETITION:**

It is well known that regular use of beta2-agonist could lead to development of tolerance to bronchodilation (i.e. reduced bronchodilator response during acute asthma) and bronchoprotection (i.e. reduced ability to prevent exercise-induced bronchoconstriction). Tolerance to bronchodilator develops rapidly, after only few doses, and regardless of ongoing treatment with inhaled corticosteroid. Tolerance to bronchoprotection develops after few weeks of treatment and regardless of ongoing treatment with inhaled corticosteroids. The decreased bronchoprotection might result in increased use of beta2-

**III SUBSTANCES PROHIBITED IN PARTICULAR SPORTS:**

Table 1. The 2011 Prohibited List by World Anti-Doping Agency.

S2. Peptide hormones, growth factors and related substances

#### **5.1 Pharmacological treatment**

Treatment of asthma and exercise-induced bronchoconstriction (EIB) in elite athletes should follow international asthma treatment guidelines like the Global Initiative for Asthma (GINA), see Figure 1 (Bateman et al 2008). The main purpose of pharmacotherapy is control of asthma symptoms, reducing airway inflammation and airway hyperresponsiveness, achieving normal lung function, and prevent exacerbations

Fig. 1. Management approach. Adapted from GINA.

#### **5.2 Beta2-agonists**

Inhaled short acting beta2-agonists (SABA), e.g. salbutamol and terbutaline, are first choice therapy for fast relief of EIB. Moreover, SABA is useful in preventing EIB if taken 15 minutes before exercise. Frequent and increased use indicates uncontrolled asthma and should result in reassessment of treatment strategies. Side effects are tremor, tachycardia, palpitations and headache, which increase in frequency and intensity with higher doses. With high systemic doses hypopotassemia and muscle convulsions can occur. Systemic use of beta2-agonists by elite athletes is entirely prohibited according to the Prohibited List, see Table 1.

Inhaled long acting beta2-agonists (LABA), e.g. salmeterol and formoterol, are used in management of uncontrolled asthma treated with inhaled corticosteroid alone. LABA is used as add-on therapy to inhaled corticosteroids, either as fixed combination or in two separate devices. LABA should never be used as monotherapy in asthma due to risk of serious adverse events with increased risk of mortality in case of exacerbation. In a review from 2005 it was concluded that combined fluticasone and salmeterol was superior to fluticasone as monotherapy in preventing EIA. Furthermore, in a randomized, doubleblinded study combined budesonide/formoterol was compared with budesonide alone, asthma control was better with reduced symptoms when treated with combination of budesonide/formoterol. A new study from 2010 confirms previous findings with more efficacy when inhaled budesonide is combined with formoterol among adults and adolescents with moderate to severe asthma. These findings indicate that it is not an effect related to the specific drug, but a class effect of the combination.

#### **I SUBSTANCES AND METHODS PROHIBITED AT ALL TIMES (IN- AND OUT-OF-COMPETITION):**

548 Allergic Diseases – Highlights in the Clinic, Mechanisms and Treatment

Treatment of asthma and exercise-induced bronchoconstriction (EIB) in elite athletes should follow international asthma treatment guidelines like the Global Initiative for Asthma (GINA), see Figure 1 (Bateman et al 2008). The main purpose of pharmacotherapy is control of asthma symptoms, reducing airway inflammation and airway hyperresponsiveness,

**and Environmental**

**Select one Select one Select one Select one**

**control**

**Medium or high-dose inhaled**

**corticosteroid + Long acting beta2-agonist**

**Leukotriene modifier**

**Theophylline**

**Oral**

**glucocorticosteroid**

**Anti-IgE treatment**

**Step1 Step2 Step3 Step4 Step5**

**Short-acting beta2-agonist as needed**

**Low-dose inhaled corticosteroid + Long acting beta2-agonist**

**Medium or highdose inhaled corticosteroid**

**corticosteroid + Leukotriene modifier**

**corticosteroid + Theophylline**

elite athletes is entirely prohibited according to the Prohibited List, see Table 1.

Inhaled short acting beta2-agonists (SABA), e.g. salbutamol and terbutaline, are first choice therapy for fast relief of EIB. Moreover, SABA is useful in preventing EIB if taken 15 minutes before exercise. Frequent and increased use indicates uncontrolled asthma and should result in reassessment of treatment strategies. Side effects are tremor, tachycardia, palpitations and headache, which increase in frequency and intensity with higher doses. With high systemic doses hypopotassemia and muscle convulsions can occur. Systemic use of beta2-agonists by

**inhaled**

**5.1 Pharmacological treatment**

**Asthma education**

**inhaled corticosteroid**

**modifier**

 **Low-dose**

 **Low-dose inhaled**

Fig. 1. Management approach. Adapted from GINA.

**Short-acting beta2-agonist as needed**

 **Low-dose**

 **Leukotriene**

**5.2 Beta2-agonists**

achieving normal lung function, and prevent exacerbations

#### **II SUBSTANCES AND METHODS PROHIBITED IN-COMPETITION:**

S0-5 and M1-3 defined above

#### **III SUBSTANCES PROHIBITED IN PARTICULAR SPORTS:**

Table 1. The 2011 Prohibited List by World Anti-Doping Agency.

It is well known that regular use of beta2-agonist could lead to development of tolerance to bronchodilation (i.e. reduced bronchodilator response during acute asthma) and bronchoprotection (i.e. reduced ability to prevent exercise-induced bronchoconstriction). Tolerance to bronchodilator develops rapidly, after only few doses, and regardless of ongoing treatment with inhaled corticosteroid. Tolerance to bronchoprotection develops after few weeks of treatment and regardless of ongoing treatment with inhaled corticosteroids. The decreased bronchoprotection might result in increased use of beta2-

Pharmaceutical Treatment of Asthma Symptoms in Elite Athletes – Doping or Therapy 551

EIA without itself inducing significant bronchoconstriction. The protective effect of different types of warm-up has been compared in controlled studies, but no consensus is found. Different study protocols and designs make it difficult to compare the existing studies, but physical warm-up in some form and extent seems generally to have a protective effect on EIA, and physical warm-up should be advised for all athletes with asthma. Breathing filters may reduce EIB among those exercising in cold conditions. A study from 2000 compared response to exercise in cold air (decrease in FEV1) in nine patients with EIA when given no treatment, given premedication with a beta2-agonist, wearing a heat-and-moisture-exchanging facemask, and given both premedication and facemask. The mean maximal change in FEV1 was 27% with no treatment, 12% with facemask, 7% with premedication, and no change with the combination of premedication and facemask. Another study from 2006 examined effects of a heat exchanger mask, placebo mask, and premedication with a beta2-agonist. Five patients with EIA performed a treadmill exercise test while breathing cold air. It was concluded that a heat exchanger mask prevented cold exercise-induced fall in lung function as effectively as treatment

During the last decade some research has focused on nutritional factors, especially omega-3 polyunsaturated fatty acids' influence on airway inflammation and exercise-induced bronchoconstriction. A review from 2005 concluded that omega-3-polyunsaturated fatty acid supplementation reduces the degree of exercise-induced bronchoconstriction compared with placebo in patients with EIA. The review was based on a small number of studies with limited range of clinically important outcomes. Further controlled and well-designed