An important feature for oral allergens is their digestion‐resistance during gastrointestinal transit. For some oral allergens, digestion stability is an innate feature, whereas digestion‐labile antigens may only persist in times of impairment of the digestive system. In this review, we collect evidence from mouse and human studies that besides the inherent molecular characteristics of a food protein, the stomach function is decisive for the allergenic potential. Gastric acid levels determine the activation of gastric pepsin and also the release of pancreatic enzymes. When anti‐ulcer drugs inhibit or neutralize gastric acid, they allow persistence of intact food allergens and protein‐bound oral drugs with enhanced capacity to sensitize and elicit allergic reactions via the oral route. Mouse studies further suggest that maternal food allergy arising from co‐application of a food protein with anti‐acid drugs results in a Th2‐biased immune response in the offspring. Especially, anti‐ulcer drugs containing aluminum compounds act as Th2 adjuvants. Proton pump inhibitors act on proton secretion but also on expression of the morphogen Sonic hedgehog, which has been related to the development of atrophic gastritis. On the other hand, atrophic gastritis and resulting hypoacidity have previously been correlated with enhanced sensitization risk to food allergens in elderly patients. In summary, impairment of gastric function is a documented risk factor for sensitization against oral proteins and drugs.

Abbreviations

GERD gastro‐esophageal reflux disease H2RA H2‐receptor antagonist HP Helicobacter pylori LOAEL lowest observed adverse effect level NOAEL no observed adverse effect level PPI proton pump inhibitor

Food allergies and intolerances – the basics Approximately 1.8–3.3% of the young adult population (aged 18–39 years) in industrialized countries like Denmark (1), France (2), or Germany (3) suffer from immunologically mediated adverse reactions to food, i.e. food allergies. However, a recent study of the European Community Respiratory Health Survey (ECRHS) found even higher numbers, with positive allergen‐specific IgE to any of 24 tested food allergens in 7–24% of a young adult population aged from 20 to 39 years in different countries of Western Europe, the USA, and Australia (4). Furthermore, 6–8% of children younger than 3 years are affected in the USA (5, 6). In these patients, sensitization occurs in a ‘silent way’, i.e. without symptoms, and leads to IgE formation and specific hypersensitivity against the offending food. There are at least two possibilities of sensitization: (i) indirectly, when respiratory allergens lead to induction of IgE that is cross‐reactive with food proteins, or (ii) directly, when oral sensitization and IgE induction occur via the gastrointestinal mucosa. As a consequence, effector cells like mast cells and eosinophils get armed by IgE, thus ready to release mediators when the relevant food allergen is ingested subsequently. This may lead to mild up to severe immediate‐type symptoms. For yet unknown reasons, the same patient may react toward food allergens to different extents at different time points of life. Today, consequent avoidance of allergen exposure is the only option for food‐allergic patients because no causative therapies are available yet. Therefore, much focus is given on the development of methods to predict allergenicity of proteins or to trace allergens in food. A multidisciplinary International Workshop held in May 2007 focused on risk assessment and safety assessment for allergenic foods and concluded that three possible approaches should be considered to protect consumers from potentially hazardous reactions: (i) safety assessment using no observed adverse effect level (NOAEL)/lowest observed adverse effect level (LOAEL) and uncertainty factors; (ii) safety assessment using Benchmark Dose and Margin of Exposure (MoE); and (iii) risk assessment using probabilistic models (7). Regarding the in vitro safety assessment, different food processing methods have been recognized to result in different impacts on stability and/or allergenicity of different foods [reviewed in (8)]. Some processes were shown to enhance either the IgE‐binding capacity, the digestion stability, or the skin prick test reactivity to food proteins, for instance roasting of peanuts (9); pasteurization of milk (10); or smoking of fish (11). However, there are also studies showing that some treatments of food can reduce its capacity to induce allergic reactions, for instance the simple addition of vinegar to extracts of boiled chicken meat, eggs, or lentils decreased their capacity to induce positive reactions in skin prick test and oral provocation test, as well as IgE‐binding in immunoblotting experiments, indicating that some allergens may already be disarmed by low pH (12). Also, technological treatment like homogenization of bovine or ovine meat can reduce the skin prick test reactivity in allergic patients (13). Additionally, formation of aggregates or multimers may influence the immunogenicity/allergenicity of antigens (14). The occurrence of dimers and/or multimers was shown to enhance immunogenicity as well as allergenicity of aeroallergens (14) and food allergens (10). Multimers can also arise during cross‐linking processes, accomplished for instance by the enzyme tissue transglutaminase in the gastrointestinal tract, which gets activated under stress‐like conditions like exercise, inflammation, etc. This cross‐linking process involves glutamine residues, for instance present in gliadins of wheat, and leads to enhanced IgE‐binding and skin prick test reactivity to the high‐molecular weight complexes formed (15). Furthermore, the food matrix might play a role in the determination of allergenicity, as for instance most allergic patients tolerate egg and milk, respectively, when heated/baked with wheat (16, 17). Another factor determining the allergenicity outcome could be the fat content of the food stuff, as a higher fat content of the vehicle also led to tolerance of higher allergen amounts in peanut‐provoked patients (18). Similarly, the presence of different plant polysaccharides used in food industry, such as xylan, pectin, and gum arabicum, was shown to change/reduce the IgE‐binding capacity of digested peanut proteins (19) or heated milk‐protein beta‐lactoglobulin (20). The search for common food allergen characteristics is, however, unsatisfactory yet, even if a limited number of proteins seem to be relevant for sensitization (21, 22): only eight foods/food groups, namely crustacean, eggs, fish, milk, peanuts, soybeans, tree nuts, and wheat account for 90% of food‐allergic reactions (23). Accordingly, in the above‐mentioned ECRHS study of young adults, the most frequent sensitizers were found to be hazelnut, peach, shrimp, and wheat (4). Importantly, this top list still remained true when birch pollen positive patients were excluded, which means that the high number of reactions to hazelnut and peach (cross‐reactive food allergens to birch pollen) cannot solely present cross‐reactive food intolerance. In contrast, no wheat‐sensitized individuals remained when the group of rye grass allergic patients was excluded in the Australian cohort of this study. Several important questions still remain open: Which are the first cells responsible for sensitization to oral allergens and turning on the IgE program? Why are some persons going to develop allergy, independent of atopic or nonatopic genetic background, whereas others being exposed toward the same type of food will not? Which factors determine the threshold of food allergens leading to symptoms in the same patient at different time points? Why are some proteins able to sensitize via the oral route although they are obviously easily digested by gastric proteases?

Physiological fate of food proteins during digestion Resistance to gastrointestinal digestion has been proposed as one dominant feature of food allergens (24). This cognition led to the development of so‐called in vitro digestion assays using simulated gastric fluid as part of food safety tests (25). Some working groups have tried to come closer to the in vivo situation by combining gastric and intestinal digestion in in vitro models (26, 27); by imitating and comparing the adult vs the infant digestion situation in vitro (26); or by using so‐called dynamic models of digestion (28), which mimic not only the influence of digestive enzymes, but also the physical processes and temporal changes in conditions of the stomach and the lumen of the small intestine, like hydration, mixing, shear forces, transport, and delivery [reviewed in (27)]. However, resistance to gastrointestinal digestion is not an innate characteristic to all food proteins, and some authors have even concluded that missing stability to digestion may speak for a minor importance of proteins as allergens. Examples are glycinin and Ara h 3 from soybean and peanut, respectively, of which the authors concluded that due to their sensitivity to peptic digestion, they may not be such important food allergens (29), although these proteins were previously described as major allergens in these legumes (30, 31). The hindrance of normal digestion of these allergens because of some external influence factors, however, has not been implicated in their allergenicity. From a physiological point of view, the gastric digestive system uses pH shifts to control the activity state of proteases and as a means to protect the mucosa against autodigestion. Gastric propepsin is activated to become pepsin by cleavage of a peptide, which is produced exclusively at low pH (32, 33). Hunger, appetite, or ingestion enhance gastric acid production from parietal cells in the stomach and lead to propepsin release from gastric chief cells, protease activation, and finally peptic protein digestion. Further transit of the acidic chymus to the duodenum leads to stimulation of the pancreas to release alkaline secrets containing the classical pancreas proteases trypsin, chymotrypsin, and carboxypeptidases. However, this releasing process is dependent on the activation of secretin, a hormone detected in 1902 by Bayliss and Starling (34). This substance is synthesized in cytoplasmic secretory granules of S‐cells, especially present in the mucosa of the duodenum and less numerous in the first portion of the jejunum (35). Importantly, the release of secretin into plasma and/or the lumen of the intestine is dependent on low duodenal pH (between 4 and 4.5) (36, 37). This low pH usually stems from the acidic chyme of the stomach (38), although digested products of fat and protein, bile acids, and some herbal extracts have also been shown to release secretin (39). There has been some controversy on this dependency of secretin release on low pH in early times after secretin detection (40); however, according to more recent work, actually the acid in the duodenum appears to be the major stimulant of secretin release (39, 41). This means that low gastric acid pH is crucial for activation and liberation of gastric and pancreatic proteases. In concert, these enzymes degrade proteins to small nutritionally valuable peptones and peptides, which optimally are ignored by the immune system or lead to tolerance. This implicates that the functional acidic stomach has a protective function against many digestion‐labile proteins, which otherwise could present antigens or even allergens. The low pH in the gastrointestinal tract is also important to minimize the risk of intestinal infections (42, 43). In line with this, it has been observed that bactericidal activity in the stomach is given when the pH is below 3.0, in contrast, bacterial overgrowth (e.g. Clostridium difficile, Campylobacter jejuni, Salmonella spp.) occurs during hypochlorhydria at pH above 4.0 (43-45). The consequence of this colonization due to breakdown of the ‘gastric bactericidal barrier’ (42, 45) is diarrhea, and actually this symptom was found to be associated with the usage of proton pump inhibitor (PPI) as the second‐most important reason after the usage of antibiotics (46). Vice versa, babies aged between 1–12 months with chronic diarrhea presented with increased gastric pH (mean pH above 4.0 in about 57%) (47). However, to complete the picture, one has to mention that also breast‐fed babies were shown to have an increased gastric pH (mean pH around 4.0), but there was no bacterial overgrowth of gram‐negative bacilli and no diarrhea, an observation that may be related to the transfer of protective factors (e.g. lysozyme, lactoferrin, IgA antibodies) by the mother’s milk. Underlining the bactericidal barrier function of the stomach, in young gastro‐esophageal reflux disease (GERD)‐affected children, aged 4–36 months, who were treated either with ranitidine [H2‐receptor antagonist (H2RA)] or omeprazole (PPI) for 8 weeks, an enhanced risk for acute gastroenteritis and community‐acquired pneumonia was detected, although children were healthy otherwise (42). These risks even sustained after ending the therapy.

Physiological situations and pathophysiological events leading to impaired digestion One population group who often presents with enhanced gastric pH (pH > 4.0 for 80–90% of time) are preterm born infants, the acidity in these children only rising with age (48). In full‐term infants, the acidity in the stomach changes during the first hours and days of life: starting with a median pH of about 6.1, it decreases to a median pH of 2.2 after 6 h of age (49). However, after this initial low pH, there occurs a loss of acidity for 10 day (pH increases), and thereafter pH only slightly goes down again (50). Adult values of gastric pH, as well as the full digestive capacity and the complete mucosal barrier function, are reached at an age of approximately 2 years only [reviewed in (51)]. For example, the output of pepsin/kilogram body weight per hour was found to be 1/15 of the adult levels on the first day of life (52) and would only be roughly comparable to that of adults in the second year of life (53). Similarly, the mean acid output in 21‐month‐old children after histamine stimulation was found to be only 50% of that observed in adults and is roughly similar to adult levels only by the end of the second year of life (54, 55). Therefore, peptic digestion may not be complete during early life, and protein remnants of the diet could act as allergens. Together, these facts may contribute to the higher incidences of food allergies in children. Food allergies in adults have been observed at any age, and thus novel sensitization principally may occur at any point of time. There are different scenarios when the stomach’s acid and pepsin production and thus protective function could be disturbed in adults. Hypochlorhydria or achlorhydria can be due to chronic atrophic gastritis. Interestingly, the other way round, it was shown that the loss of parietal cell function is preceding the development of gastric atrophy. Owing to parietal cell loss, hydrochloric acid is missing, and this results in the negative fate of other cell lineages in the stomach leading to atrophic gastritis (56). In parallel, the expression of the morphogen called Sonic hedgehog (Shh) correlated with atrophic gastritis and metaplasia in the intestine (57). It was further shown that Shh expression and signaling depends on gastrin levels and gastric acidity, as the omeprazole‐induced hypoacidity inhibited the cleavage of the precursor protein of Shh. This cleavage is mediated by the protease pepsin A, which is acid‐dependent (58). Omeprazole furthermore reduced the expression of Shh mRNA (59). Loss of Shh expression, however, is not the outcome of loss of glandular cells but is preceding it, although not necessarily being causative for parietal cell loss. Nevertheless, loss of Shh may be involved in hypochlorhydria and hypergastrinemia with subsequent morphological changes and loss of sensitivity to histamine‐regulated acid secretion (60). Summing up these recent data, Shh is responsible for parietal cell function, the loss of it leads to hypochlorhydria and hypergastrinemia, resulting in atrophic gastritis (61). The circle closes as Shh is acid‐dependent and therefore could be reduced/inhibited by acid‐suppressing drugs. Based on these facts, we hypothesize that the treatment for atrophic gastritis with acid‐suppressing drugs (see below) may in fact accelerate the disease. Actually, studies have shown that all patients on long‐term treatment with PPIs develop hypergastrinemia (as is the case during any situation with lowered gastric acid secretion) (62); however, other authors have attributed the main reason for atrophic gastritis to Helicobacter pylori (HP) infection and dismissed the contribution of PPIs (63). Achlorhydria may be frequently found in older people (32.4% in subjects aged 74–80 years) (64); in context with chronic alcohol consumption (65); or during acute (66, 67); or chronic gastric infection (68). In addition, low gastric acidity, for instance due to atrophic gastritis, represents an enhanced risk for oral infection e.g. elderly people are more likely to be infected with HP (69). Other diseases with reduced acid production are chronic renal failure (70) and gastric ulcer (in contrast to duodenal ulcer), where patients may present with decreased basal and stimulated acid production (71). Also, resections and bypasses of the stomach, including bariatric surgery, cause achlorhydria (72). More importantly, inhibition or neutralization of gastric acid with so‐called anti‐ulcer drugs is performed when treating gastritis and peptic ulcers. The therapy goal is to reach gastric pH levels above 4.5 (73), also for children (74), or even above 6.0 for treating bleeding peptic ulcers (75). Avoidance of acid is needed in these therapies to stop autodigestive processes and support mucosal healing in the extreme environment of the gastric lumen. Long‐term treatments with steroidal and nonsteroidal anti‐inflammatory drugs are associated with gastric mucosal damage for various pharmacological reasons. Therefore, anti‐ulcer drugs are also co‐prescribed during these treatments to protect the gastric mucosa. Several classes of medications are available for these purposes: PPIs already mentioned above; H2RA; sucralfate; and acid neutralizers like bismuth compounds. Our in vitro digestion assays have indicated that already at pH 2.75–3.0 pepsin is no longer fully activated (76, 77). The work of Tanaka and colleagues also underlines this observation, as the activation of pepsins optimally occurs at pH 1.0–3.5 and at higher pH drops significantly (78, 79). As a consequence, peptic digestion is significantly hampered. This implies that during medications with anti‐ulcer drugs, when pH values even get much higher (80), also digestion‐labile food proteins might acquire allergenic potency (Fig. 1). Figure 1 Open in figure viewer PowerPoint The fate of a food protein depends on its intrinsic stability and/or the gastric acidity level at the time point of ingestion. (A) Proteins homologous to Bet v 1 from birch pollen are easily degraded. They cause oral, but rarely systemic reactions because in degraded form they are ignored or tolerated by the immune system. (B) Digestion‐labile proteins, like parvalbumin from codfish, may become allergenic when digestion is inhibited. This may occur in conditions where the gastric pH is elevated, resulting in diminished activation of pepsin. The elevated pH of the chyme subsequently prevents the release of duodenal secretin resulting in impaired pancreatic digestive function. Persisting protein remnants are then able to induce an (allergic) immune response. (C) Sensitization against digestion‐stable food proteins/oral antigens like Ara h 2 occurs independently of the gastric acid condition because of specific molecular features causing stable tertiary or quaternary structures of these proteins.

From a case of caviar allergy to a novel mouse model of food allergy Our work was originally triggered by a case of Beluga caviar allergy (81) occurring in a patient suffering from chronic ulcer and constantly taking PPIs during years. The patient noted specific sensitization several years after the first caviar consumption when experiencing anaphylactic episodes. As caviar consumption usually is a rare event, he remembered that the first time eating caviar happened during treatments with PPIs. We imitated this case in an animal study and fed mice with caviar (82). Only those groups that were additionally treated with sucralfate, H2 receptor blockers, or PPIs developed an allergic phenotype consisting of specific IgE and positive immediate‐type skin tests. Subsequently, we repeated the experiments with other types of allergens, like parvalbumin from fish (82), hazelnut (83), and recently ovalbumin (84) and peanut (unpublished). In these novel animal models of food allergy, we also found dense infiltration with eosinophils in the gastric mucosa (85), which may support IgE production via IL‐4 secretion. Furthermore, the morphology and immunologic setting of the gastrointestinal tract was altered in animals that received sucralfate along with the allergen, as this treatment induced changes in the structure of epithelium and villi, and an increase in eosinophils and mucus‐producing cells in the intestine (86).

Anti‐ulcer medications are relevant in human patients To evaluate the impact of our observations for human patients, we designed an observational study of 150 gastroenterological patients who were treated during 3 months with anti‐ulcer drugs. The data showed that anti‐ulcer drugs indeed supported IgE induction and de novo sensitization against food proteins from the average daily diet (87). The sensitization was clinically relevant and long lasting, as skin prick tests toward specific food allergens remained mostly positive even 5 months after discontinuation of anti‐ulcer therapy. As the patients were around 65 years of age, the results indicated that oral sensitization may occur also in elderly persons. Moreover, we concluded that by the oral route also established tolerance toward previously well‐tolerated food can be broken. Another important target group for anti‐ulcer drugs are pregnant women. During pregnancy, anti‐ulcer drugs are consumed because of heartburn/gastro‐esophageal reflux (88), which is caused by hormonal changes leading to reduced esophageal sphincter pressure (89), and by increasing abdominal volume. Whereas in the past modifications of life‐style and short‐term treatments with acid neutralizers were preferred (88), also this patient group is treated with highly efficient PPIs today (90). We hypothesized that in this setting, sensitization of the mother could predispose the child for Th2 immune responses. When we fed pregnant mice with fish protein in context with anti‐ulcer medication, we observed not only allergy induced in the mother animal but also a Th2 bias in the offspring (91). This prompted us to suggest that these treatments in pregnancy might contribute to the increased numbers of atopic predisposition in babies (92). Interestingly, when Dehlink et al. investigated three data sets from a Swedish birth registry and correlated the allergy/asthma incidences of babies with anti‐ulcer drug consumption of the mothers, they could confirm our prediction in the human setting (93).

Sucralfate with an aluminum compound acts as immunostimulant In immunology, aluminum compounds such as Al(OH) 3 are classically used as Th2 adjuvants. Sucralfate is a hydrous basic aluminum salt of sucrose octasulfate. Indeed, its adjuvant effect could be recently demonstrated in a mouse model (94, 95), where ovalbumin as a less degradable protein was used as a model allergen and applied together with sucralfate. The induced allergen‐specific IgE‐antibodies as well as IL‐4 and IL‐5 cytokines indicated a Th2 shift when sucralfate was co‐applied. Therefore, whereas other antacids and the dietary supplement base powder bind and reduce acid only (76), sucralfate additionally acts as immune stimulant (94). It was further confirmed in a recent study by another working group that aluminum does also impact the human immune response, as the addition of aluminum hydroxide to the model antigen keyhole limpet hemocyanin (KLH) resulted in a clear Th2‐response (96) compared with studies where KLH was used without an adjuvant (97).

Impaired digestion lowers threshold levels of food allergens In the sensitization phase of allergy, IgE‐antibodies are obviously formed against conformational epitopes but not against smaller peptides, except in persistent allergy. Does this also apply for the effector phase when IgE is already formed and fixed to effector cells? We hypothesized that conformationally intact allergens would trigger mediator release much easier, whereas peptide leftovers should no longer be able to crosslink IgE, which would have implications in clinics. Indeed, when in a clinical study, fish allergic patients were orally exposed to native or in vitro predigested fish, the native protein expressed much higher capacity to elicit clinical reactivity and positive type I skin test reactions (98). Moreover, in histamine release test, the dose of native allergen eliciting positive reactivity was 10 000 times lower than with predigested allergen (77). This implies that in settings of impaired digestion, lower levels of allergens may be able to induce hypersensitivity reactions. These data might finally also explain why some food‐allergic patients develop symptoms of different intensity at different time points: their actual symptom intensity may depend on the current functional capacity of the digestive system. We consider this observation being clinically important and suggest that a question clarifying anti‐ulcer drug consumption should be regularly included in allergologic anamnesis.

Anti‐ulcer drugs as co‐medications: role in drug allergy? Anti‐ulcer drugs are useful co‐medications to protect the mucosa from damage due to steroidal and nonsteroidal drugs. This is especially relevant during long‐termpain‐ and anti‐inflammatory therapies, for instance in management of rheumatoid arthritis or inflammatory bowel disease. A number of these patients develop intolerances to these drugs. It is apparent that in some incidences, clinical symptoms are of the immediate‐type and mimic antibody‐mediated hypersensitivity. However, serum tests often remain negative for specific IgE or IgG possibly due to methodological limitations. Moreover, these drugs are haptens and should act as complete antigens only in context with other proteins. In studies of the pharmacokinetics of oral drugs, the adsorption to gastric proteins hindering regular distribution is a well‐known phenomenon. Especially, albumin occurring at high levels in gastric juice is a preferred binding partner for oral drugs, for instance for the pain killer diclofenac. Thereby, a complete antigen is formed as an intermediate product (99). However, this product is normally digested by pepsin, leading to the release of the drug. It was tempting to speculate that a problem might arise when anti‐ulcer drugs are co‐medicated. Exactly, in this setting, the intermediate drug‐carrier complexes would possibly be able to persist the transit and sensitize the patient. Could this be an explanation for antibody‐mediated drug allergy? The answer was found in a BALB/c mouse model when a drug‐albumin conjugate was fed with and without co‐medication of the anti‐ulcer drugs sucralfate and PPI (100). Only the group treated with anti‐acid drugs developed IgE and IgG1 against diclofenac. Clearly, more studies are needed to confirm this theory in the human setting.

Synopsis Based on in vitro data as well as mouse and human studies, we propose that anti‐ulcer drugs support clinically relevant sensitization against oral proteins like food allergens and drugs and possibly favor eosinophilic gastrointestinal inflammation (101). The induced Th2 bias is long lasting, clinically relevant and transmitted to the offspring during pregnancy. Several immunological mechanisms may be causative: (i) anti‐ulcer drugs inhibit peptic digestion and turn harmless food proteins into allergenic molecules, which are able to perform sensitization and to elicit allergy at lower threshold levels and (ii) some anti‐ulcer compounds such as sucralfate exhibit additional Th2 adjuvant function. The patient groups affected are those treated or co‐medicated with anti‐ulcer drugs to heal or prevent gastro‐esophageal reflux, gastritis, or gastric ulcer. Moreover, a high number of patients use anti‐acid substances, antacids, or dietary supplements – without a prescription of a physician – on a more or less daily basis to treat heartburn and stomach ache. Acid‐suppressing drugs are considered safe, with – admittedly rare‐ side effects [reviewed in (71) and (102)] (Table 1). However, to this list, we suggest that ‘Increased risk for sensitization against dietary proteins’ and ‘Lowering the food allergens levels needed to elicit hypersensitivity reactions in food‐allergic patients’ should be added. Therefore, we question over‐the‐counter sale of anti‐ulcer drugs and suggest prescribing them according to strict indications during a therapeutically useful period of time, especially during pregnancy. Table 1. Undesired effects of different acid‐suppressing drugs* Drug/mechanism of drug Side effects Antacids/neutralize produced acid Diarrhea Constipation Interference with drug absorption Renal, metabolic, and acid‐base disturbances H2‐receptor antagonists/block histamine receptor 2 and therefore histamine‐induced acid production Mental confusion Interference with drug absorption Gynecomastia Interstitial nephritis Cytochrome P450 interactions Proton pump inhibitors/block H+K+‐ATPase and therefore complete acid production during their presence Hypergastrinemia Rebound acid hypersecretion Malabsorption Infections Drug interactions

Acknowledgments Financial support has been obtained by the Hertha Firnberg stipend T283‐B13 and grant SFB1808‐B13 of the Austrian Science Fund (FWF).