Masters Research - Masters of Medical Science / The goal of this thesis is to report on research that explored the role of cow’s milk protein in children with chronic functional constipation. The research consisted of a systematic review of the literature, two clinical crossover trials, and a qualitative exploration of the lived experience of following a milk-free diet. Chapter 1 provides the introduction to both allergy and constipation, and the relationship between the two. Causes of constipation can be organic or functional (1). Organic causes of constipation occur in relation to a primary disease classification such as endocrine or metabolic disorders, neurologic disorders, anatomic malformation, collagen vascular disease and some drugs (for example, opiates). Chronic functional constipation is defined as having one bowel motion every three to 15 days (2) and is characterised by painful bowel movements or strain in defecation, hard stools with increased diameter or pellets, and occurs with or without soiling (3). This functional constipation is defined as chronic when it persists for greater than two weeks (4). Chapter 2 details the methods used in searching the literature for evidence for a role of cow’s milk consumption in chronic functional constipation in children from 1980 to 2006. This was published as a systematic review. The literature surrounding cow’s milk and constipation was found to be limited. None of the studies previously conducted were population-based or structured to provide evidence-based evaluation or treatment guidelines at either the general practitioner or paediatric specialist level. The strongest evidence found was a double blind randomised control trial conducted by Iacono and colleagues (3). The research study by Iacono and colleagues (3) provides evidence of an association between cow’s milk and constipation. The following research questions were developed from the systematic review: 1. Can the results of the Iacono and colleagues study of children with chronic functional constipation that respond to the replacement of cow’s milk protein with soy be replicated in the Australian setting? 2. Does cow’s milk β casein A1 cause constipation in children with chronic functional constipation? 3. What are the immunological and biochemical mechanisms underlying chronic functional constipation that respond to the removal of cow’s milk protein in children? 4. What factors affect the feasibility of parents administering a cow’s milk protein free diet to their children? The four questions were addressed by two different dietary crossover trials and a qualitative study. Chapter 3 describes the participants recruited and the methods used for the crossover trials investigating milk protein and paediatric chronic functional constipation including details of the primary outcome measure (number of bowel motions during a two-week trial period) and secondary outcome measures (biochemical, immunological and faecal analysis). Chapter 4 describes the results of Trial 1, which replicated the Iacono and colleagues study in the Australian setting, investigating the effects of soy and cow’s milk β-casein A1 in children with chronic functional constipation. One hundred percent of participants experienced resolution of their constipation during the soy milk condition compared with 68% experiencing resolution during the soy milk condition in the Iacono and colleagues study (n=65). Thirteen participants were recruited to Trial 1. Nine participants returned constipation diaries for the study period. The mean (SD) number of stools for each of the conditions was: baseline, 5.1 (1.4); cow’s milk 9.9 (4.4); washout 13.0 (5.2); and soy milk 15.1 (5.0). The differences between the three dietary conditions were statistically significant, p=0.03. The results confirmed the hypothesis that children in the Australian setting with chronic functional constipation unresponsive to the usual treatments, respond to the removal of cow’s milk protein from the diet. Chapter 5 describes the results of Trial 2, the double blind crossover trial comparing the effects of cow’s milk β-casein A1 and cow’s milk β-casein A2 in children with chronic functional constipation. Thirty-nine participants were recruited to Trial 2 and 26 participants returned constipation diaries for the trial period. Unlike the soy result, the cow’s milk β casein A2 did not give 100% resolution of constipation, in fact, the percentage resolution was almost identical to the cow’s milk β casein A1 result. The fact that some children responded during the cow’s milk casein A1 condition in both trials could be caused by a threshold effect, given it was likely that participants were consuming less cow’s milk protein during the trial (400 mL with elimination of all other sources of cow’s milk protein) than on their pre-trial diet. Resolution with both the cow’s milk β casein A1 and cow’s milk β casein A2 conditions suggests that these children are able to tolerate some cow’s milk protein before the symptom of constipation occurs. This could be a food intolerance type reaction or there is some other component in cow’s milk that is causing the problem in these children. Chapter 6 describes a qualitative study of the feasibility for mothers to administer a cow’s milk protein free diet to their children. The experiences of mothers following a cow’s milk protein free diet to assist in the management of chronic functional constipation in children were reported. A number of themes were identified that are useful to health professionals educating families. Mothers found the removal of cow’s milk protein from the diets of their children challenging but persevered due to the potential benefit to their children. Many mothers planned to continue post study with a modified approach to the cow’s milk protein free diet by allowing some cow’s milk protein in the diet to make the diet more acceptable to the family but not as much as the pre-trial diet. These experiences provide health professionals with valuable insights and ideas to assist their patients to manage a cow’s milk protein free diet. Chapter 7 discusses all aspects of the research including any limitations. The results of Trial 1 confirmed the hypothesis that children in the Australian settling with chronic functional constipation unresponsive to the usual treatments respond to the removal of cow’s milk protein from the diet. Therefore, cow’s milk protein is involved in the aetiology of constipation in these children. All the study participants demonstrated an absence or low level of normal gut flora, which may affect bowel regularity. Further research into species present and absent may provide further explanations to the lack of bowel regularity in these children. The immunological and biochemical mechanisms underlying chronic functional constipation that respond to the removal of cow’s milk protein requires further investigation. Although the number of statistically significant variables between the conditions was low, there was a high degree of abnormality. Further investigations are needed, including research into food intolerance reactions that affect the nerve endings in the bowel. The results in Trial 1 and Trial 2 are suggestive of an involvement of blood factors including platelets and monocytes. Other children may have a chronic Streptococcus A infection which may be contributing to constipation as well as to liver function abnormalities. Liver function abnormalities were observed for some participants in both trials, independent of milk condition. The extent to which the research questions have been answered is evaluated in Chapter 7, which includes the conclusions and recommendations of this research. In brief, the findings were: • Children with chronic functional constipation that is unresponsive to the traditional treatments should trial a cow’s milk protein free diet for at least two weeks to determine whether this may resolve the constipation. During this period, the numbers and form of bowel motions should be recorded and results compared to a one week record collected prior to commencing the cow’s milk protein free diet. • Due to the complicated nature of a cow’s milk protein free diet, especially the number of processed foods which contain hidden cow’s milk protein, consultation with a dietitian is essential for implementation of this diet. The dietitian should consider educating the patient’s family, both parents and siblings, to ensure the best outcome in terms of acceptance and compliance of the diet, and provide adequate resources. • If this dietary modification is successful for the child and alleviates constipation, consultation with a dietitian is recommended to determine the amount tolerated and nutritional adequacy of the diet. Soy milk is recommended as a substitute for cow’s milk and a probiotic needs to be prescribed to assist with the normalisation of gut flora. • Education of health professionals such as general practitioners, paediatricians, and paediatric continence nurses, regarding a cow’s milk protein free diet for chronic functional constipation, is essential to support the child and his/her family and integral to the success of this strategy. The findings of this research will be published in the scientific literature and as conference presentations. It is hoped that these findings will assist in the management of children with chronic functional constipation unresponsive to the traditional treatments.

Masters Research - Masters of Medical Science / The goal of this thesis is to report on research that explored the role of cow’s milk protein in children with chronic functional constipation. The research consisted of a systematic review of the literature, two clinical crossover trials, and a qualitative exploration of the lived experience of following a milk-free diet. Chapter 1 provides the introduction to both allergy and constipation, and the relationship between the two. Causes of constipation can be organic or functional (1). Organic causes of constipation occur in relation to a primary disease classification such as endocrine or metabolic disorders, neurologic disorders, anatomic malformation, collagen vascular disease and some drugs (for example, opiates). Chronic functional constipation is defined as having one bowel motion every three to 15 days (2) and is characterised by painful bowel movements or strain in defecation, hard stools with increased diameter or pellets, and occurs with or without soiling (3). This functional constipation is defined as chronic when it persists for greater than two weeks (4). Chapter 2 details the methods used in searching the literature for evidence for a role of cow’s milk consumption in chronic functional constipation in children from 1980 to 2006. This was published as a systematic review. The literature surrounding cow’s milk and constipation was found to be limited. None of the studies previously conducted were population-based or structured to provide evidence-based evaluation or treatment guidelines at either the general practitioner or paediatric specialist level. The strongest evidence found was a double blind randomised control trial conducted by Iacono and colleagues (3). The research study by Iacono and colleagues (3) provides evidence of an association between cow’s milk and constipation. The following research questions were developed from the systematic review: 1. Can the results of the Iacono and colleagues study of children with chronic functional constipation that respond to the replacement of cow’s milk protein with soy be replicated in the Australian setting? 2. Does cow’s milk β casein A1 cause constipation in children with chronic functional constipation? 3. What are the immunological and biochemical mechanisms underlying chronic functional constipation that respond to the removal of cow’s milk protein in children? 4. What factors affect the feasibility of parents administering a cow’s milk protein free diet to their children? The four questions were addressed by two different dietary crossover trials and a qualitative study. Chapter 3 describes the participants recruited and the methods used for the crossover trials investigating milk protein and paediatric chronic functional constipation including details of the primary outcome measure (number of bowel motions during a two-week trial period) and secondary outcome measures (biochemical, immunological and faecal analysis). Chapter 4 describes the results of Trial 1, which replicated the Iacono and colleagues study in the Australian setting, investigating the effects of soy and cow’s milk β-casein A1 in children with chronic functional constipation. One hundred percent of participants experienced resolution of their constipation during the soy milk condition compared with 68% experiencing resolution during the soy milk condition in the Iacono and colleagues study (n=65). Thirteen participants were recruited to Trial 1. Nine participants returned constipation diaries for the study period. The mean (SD) number of stools for each of the conditions was: baseline, 5.1 (1.4); cow’s milk 9.9 (4.4); washout 13.0 (5.2); and soy milk 15.1 (5.0). The differences between the three dietary conditions were statistically significant, p=0.03. The results confirmed the hypothesis that children in the Australian setting with chronic functional constipation unresponsive to the usual treatments, respond to the removal of cow’s milk protein from the diet. Chapter 5 describes the results of Trial 2, the double blind crossover trial comparing the effects of cow’s milk β-casein A1 and cow’s milk β-casein A2 in children with chronic functional constipation. Thirty-nine participants were recruited to Trial 2 and 26 participants returned constipation diaries for the trial period. Unlike the soy result, the cow’s milk β casein A2 did not give 100% resolution of constipation, in fact, the percentage resolution was almost identical to the cow’s milk β casein A1 result. The fact that some children responded during the cow’s milk casein A1 condition in both trials could be caused by a threshold effect, given it was likely that participants were consuming less cow’s milk protein during the trial (400 mL with elimination of all other sources of cow’s milk protein) than on their pre-trial diet. Resolution with both the cow’s milk β casein A1 and cow’s milk β casein A2 conditions suggests that these children are able to tolerate some cow’s milk protein before the symptom of constipation occurs. This could be a food intolerance type reaction or there is some other component in cow’s milk that is causing the problem in these children. Chapter 6 describes a qualitative study of the feasibility for mothers to administer a cow’s milk protein free diet to their children. The experiences of mothers following a cow’s milk protein free diet to assist in the management of chronic functional constipation in children were reported. A number of themes were identified that are useful to health professionals educating families. Mothers found the removal of cow’s milk protein from the diets of their children challenging but persevered due to the potential benefit to their children. Many mothers planned to continue post study with a modified approach to the cow’s milk protein free diet by allowing some cow’s milk protein in the diet to make the diet more acceptable to the family but not as much as the pre-trial diet. These experiences provide health professionals with valuable insights and ideas to assist their patients to manage a cow’s milk protein free diet. Chapter 7 discusses all aspects of the research including any limitations. The results of Trial 1 confirmed the hypothesis that children in the Australian settling with chronic functional constipation unresponsive to the usual treatments respond to the removal of cow’s milk protein from the diet. Therefore, cow’s milk protein is involved in the aetiology of constipation in these children. All the study participants demonstrated an absence or low level of normal gut flora, which may affect bowel regularity. Further research into species present and absent may provide further explanations to the lack of bowel regularity in these children. The immunological and biochemical mechanisms underlying chronic functional constipation that respond to the removal of cow’s milk protein requires further investigation. Although the number of statistically significant variables between the conditions was low, there was a high degree of abnormality. Further investigations are needed, including research into food intolerance reactions that affect the nerve endings in the bowel. The results in Trial 1 and Trial 2 are suggestive of an involvement of blood factors including platelets and monocytes. Other children may have a chronic Streptococcus A infection which may be contributing to constipation as well as to liver function abnormalities. Liver function abnormalities were observed for some participants in both trials, independent of milk condition. The extent to which the research questions have been answered is evaluated in Chapter 7, which includes the conclusions and recommendations of this research. In brief, the findings were: • Children with chronic functional constipation that is unresponsive to the traditional treatments should trial a cow’s milk protein free diet for at least two weeks to determine whether this may resolve the constipation. During this period, the numbers and form of bowel motions should be recorded and results compared to a one week record collected prior to commencing the cow’s milk protein free diet. • Due to the complicated nature of a cow’s milk protein free diet, especially the number of processed foods which contain hidden cow’s milk protein, consultation with a dietitian is essential for implementation of this diet. The dietitian should consider educating the patient’s family, both parents and siblings, to ensure the best outcome in terms of acceptance and compliance of the diet, and provide adequate resources. • If this dietary modification is successful for the child and alleviates constipation, consultation with a dietitian is recommended to determine the amount tolerated and nutritional adequacy of the diet. Soy milk is recommended as a substitute for cow’s milk and a probiotic needs to be prescribed to assist with the normalisation of gut flora. • Education of health professionals such as general practitioners, paediatricians, and paediatric continence nurses, regarding a cow’s milk protein free diet for chronic functional constipation, is essential to support the child and his/her family and integral to the success of this strategy. The findings of this research will be published in the scientific literature and as conference presentations. It is hoped that these findings will assist in the management of children with chronic functional constipation unresponsive to the traditional treatments.

Les maladies allergiques sont de plus en plus fréquentent. Elles atteignent souvent l’enfant jeune chez qui l’allergie respiratoire et l’allergie alimentaire sont les principales pathologies. L’unique traitement curatif est l’immunothérapie spécifique d’antigène (ITA), largement développée dans l’allergie respiratoire et encore à ses débuts dans l’allergie alimentaire. Pour adapter au mieux la prise en charge du patient, le diagnostic précis de l’allergie est indispensable et il n’existe actuellement pas d’examen biologique totalement fiable. Seul, la présence d’IgE spécifiques permet de diagnostiquer une sensibilisation à un allergène mais pas une allergie cliniquement symptomatique. Dans une première partie, nous avons étudié l’intérêt d’un test fonctionnel, l’ELISpot (Enzyme-linked immunosorbent spot), dans le diagnostic de l’allergie aux acariens chez l’enfant asthmatique. Le nombre de lymphocytes T circulants spécifiques d’acariens sécréteur d’interleukine (IL)-4 ou d’IL-13 était associé à la présence d’une allergie symptomatique, indépendamment des IgE spécifiques. Il était plus élevé dans le cas d’une rhinite allergique sévère et plus faible dans le cas d’une rhinite allergique légère. De plus, il variait au cours de l’année en fonction des saisons avec un pic en automne et un pic en début de printemps. Dans une deuxième partie, nous avons étudié l’intérêt de l’ELISpot dans le diagnostic de l’allergie au lait de vache chez l’enfant, confirmée par un test de provocation orale en double aveugle. Nous avons décrit que le nombre de lymphocytes T spécifiques de la caséine et sécréteurs d’IL-4 et d’IL-13 était associé à l’allergie au lait de vache avec une sensibilité de 100%. Par ailleurs, le nombre de lymphocytes T spécifiques de la caséine était également associé à la dose maximale de lait tolérée par l’enfant.Enfin, dans une troisième partie, nous avons étudié la réponse lymphocytaire T au cours d’une ITA sub-linguale (SLIT) d’une part et sous-cutanée (SCIT) d’autre part, chez des enfants asthmatiques allergiques aux acariens suivis pendant une année. Nous avons décrit une diminution des lymphocytes Th2 (sécréteurs d’IL-4 et IL-13) spécifiques d’acariens après 12 mois de SLIT associée à une augmentation des cellules sécrétrices d’IL-10 (Tr1) spécifiques d’acariens après 6 mois de SLIT. De plus, les lymphocytes T régulateurs (CD4+CD25hiCD127loFoxp3+) étaient augmentés après 12 mois de SCIT. Nous n’avons pas retrouvé de production accrue d’interféron γ (IFNγ) par les lymphocytes T spécifiques d’acariens au cours de la désensibilisation.Au total, ce travail nous a permis de décrire qu’un test fonctionnel, l’ELISpot, permet de réaliser un diagnostic fiable de l’allergie aux acariens et de l’allergie au lait de vache chez l’enfant. Par ailleurs, l’ITA induit une diminution des cellules Th2 et une augmentation des cellules Tr1 par voie sub-linguale ainsi qu’une augmentation des Treg Foxp3+ par voie sous-cutanée sans immunodéviation Th2/Th1, chez l’enfant allergique aux acariens. / Allergic diseases are steadily increasing steadily and especially in children. Allergen specific immunotherapy (desensitization) is the only curative treatment for which accurate diagnosis of allergy is essential. Currently, the presence of specific IgE diagnoses a sensitization to an allergen but not a clinically symptomatic allergy. In a first part, we studied the value of a functional test, the ELISpot (Enzyme-linked immunosorbent spot) in the diagnosis of allergy to house dust mites (HDM). The number of circulating HDM-specific IL-4 and IL-13 secreting T cells was associated with the presence of symptoms, regardless of specific IgE and was higher in severe rhinitis than in mild rhinitis. In addition, it varied according to the season with a peak in autumn and a peak in early spring (wet periods with greater allergen exposure). In a second part, we studied the value of ELISpot for the diagnosis of cow's milk allergy in children, confirmed by double blind placebo control food challenge. We found that the number of casein-specific IL-4 and IL -13 secreting T-cells was associated with allergy to cow's milk. It was also inversely correlated to the cow’s milk tolerated cumulative dose. Receiver-operating characteristic (ROC) curve of combined IL-4 and IL-13 analysis was generated. AUC was 0,98 (95% CI 0.90-1.06). For a cut-off of 10 IL-4- and 12 IL-13 secreting T-cells, sensitivity and negative predictive value were 100%.Finally, in the third part, we monitored antigen specific T-cell response in HDM allergic children treated with sublingual ITA (SLIT) on the one hand and subcutaneous ITA (SCIT) on the other hand, during one year. We found a decrease in HDM specific Th2 cells after 12 months of SLIT associated with an increase in HDM specific IL-10 secreting T-cells after 6 months of SLIT. In addition, regulatory T cells (CD4 + CD25hiCD127loFoxp3+) were increased after 12 months of SCIT. In conclusion, this work has allowed us to describe a functional test, the ELISpot, as a reliable tool for the diagnosis of mite allergy and cow's milk allergy in children. In addition, in HDM allergic children, a decrease of Th2 cells and an increase of IL-10 secreting T-cells was found in children treated with SLIT to HDM as well as an increase in Foxp3+ Treg in children treated with SCIT.