Immunoglobulin E (IgE) is an important mediator in atopic allergies. This thesis describes the development of a therapeutic vaccine against IgE and its effects in rats and dogs. The development of an assay to determine IgE levels in dogs, and the finding of a chromosome region in rats that affects IgE levels are also reported. The vaccine is a chimeric molecule consisting of the constant domains Cε2, Cε3 and Cε4 from IgE. The target domain of the vaccine is the Cε3 domain in the recipient species, which is the domain directly involved in receptor binding, while the flanking regions, Cε2 and Cε4, are from a distantly related mammal. In rats, the vaccine induced an immune response against circulating IgE, which decreased IgE levels by 90% and substantially reduced their allergic symptoms. Further, the effects of adjuvants in rats and dogs were evaluated, and when co-administered with the vaccine certain adjuvants were shown to increase the immune response against IgE. Mineral-oils were the most potent adjuvants in inducing a response against IgE, but metabolizable oils spiked with immunostimulatory substances were also efficient. It was also shown that the therapeutic vaccine could induce a decrease in IgE levels in adult dogs, even though their initial levels were exceptionally high compared with humans. The IgE levels in 76 dogs ranged between 1 and 41 μg/ml while humans normally have around 150 ng/ml. However, the high IgE levels did not correlate to any specific breed, nor did they distinguish between dogs that were diagnosed as healthy and those suffering from atopic eczema, autoimmunity or skin parasites. Regulation of total IgE levels probably involves many genes. In the final phase of the study, one candidate locus known to be involved in arthritis susceptibility in rats was investigated, and was found also to affect IgE levels.

Mast cell (MC) accumulation around tumours is an old observation gaining new relevance due to the multifaceted nature of MCs and their many roles in immunity, beyond allergy. Knowledge about tumour specific recruitment of, and interactions with, MCs is needed to unravel the function of their presence. This study investigates the participation of mast cells in the tumourigenesis of Hodgkin lymphoma (HL), a tumour with many inflammatory features. We report that MC recruitment into HL lymphomatous tissue is possibly due to the production of CCL5/RANTES by malignant Hodgkin and Reed-Sternberg (HRS) cells. In addition, increased levels of IL-9, a cytokine implicated in mast cell heterogeneity and as an autocrine growth factor for HRS cells, were found in HL patient sera and correlate with negative prognostic factors. The ubiquitous expression of CD30 by HRS cells has been implicated in HL tumour development. In HL tissue MCs were found to be the predominant CD30 ligand (CD30L) expressing cells, and through CD30L/CD30 engagement they induced a proliferative response in HRS cells. This interaction proved to be bi-directional as it induced a degranulation-independent de novo synthesis of a specific set of chemokines in MCs, including IL-8. This novel trigger of MC activation is suggested to be of importance also in atopic dermatitis (AD) and psoriasis since increased numbers of CD30L and IL-8 positive MCs were detected along with increased expression of CD30. Data presented in this study supports a specific recruitment of MCs into HL tumours and co-operative interactions between HRS cells and MCs. Our identification of reversed signalling via CD30L as a novel MC trigger provides a mechanism behind leukocyte infiltration and chronic development in diseases associated with CD30 and MCs, such as HL, AD and psoriasis.

Atopic dermatitis (AD), ichthyosis vulgaris (IV), and X-linked recessive ichthyosis (XLRI) are characterized by dry skin and impaired skin barrier. AD and IV are related to loss-of-function mutations in FLG (encoding filaggrin), whereas XLRI is caused by deletions or inactivating mutations in the steroid sulphatase gene (STS). Patients regularly use moisturizing creams, but little is known about the creams’ effects on the skin barrier. The present work combines objective scorings, non-invasive techniques, and molecular analyses of skin biopsies to characterize the skin in 57 patients with AD, IV, or XLRI, and in 14 healthy controls. Patients were classified according to their FLG and STS mutation status: AD with FLG+/+ (n = 14), AD with FLG+/– (n = 14), AD/IV with FLG–/– (n = 15), and XLRI with STS– (n = 14), as well as one man with a novel point mutation. Assessments were conducted at baseline and after four weeks of treatment with three different moisturizers applied to volar forearm skin. At baseline, dryness scoring and non-invasive assessments verified impaired skin barrier function in all patients. In patients with AD/IV, microarray analysis identified 300–3000 up- or downregulated mRNA transcripts involved in signalling pathways important for inflammation and barrier repair. The skin phenotype and number of altered transcripts were correlated with the FLG mutation status, with FLG–/– patients displaying the highest transepidermal water loss (TEWL) and the most altered transcript levels. In contrast, despite an equally dysfunctional skin barrier, only limited changes in mRNA transcripts occurred in XLRI patients. Treatment with moisturizers improved skin dryness similarly in all groups, but TEWL behaved differently: it decreased slightly in the AD/IV group and increased in the XLRI group, especially after urea treatment. Only minute effects on skin pH and mRNA expression were observed. In conclusion, FLG mutations elicit pro-inflammatory mechanisms probably aimed at restoring barrier competence. This does not occur in patients with XLRI, presumably because STS deficiency automatically increases the barrier thickness. Moisturizing treatment improves skin dryness in patients with AD, IV, or XLRI, but does not seem to normalize the altered epidermal gene expression profile in AD/IV patients.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Medical Sciences

Isolation and identification of all yeast species found on skin affected by atopic dermatitis, evaluation of their influence to the synthesis of IgE antibodies, and assessment of the possible cross-reactivity between different yeast species was performed. It was shown that in 36.9 % of the cases of atopic dermatitis, the affected skin was colonized with yeast belonging to three genera: Candida, Malassezia and Rhodotorula. Systematic and phylogenetic analysis of sequences from atypical Malassezia restricta strain M8 indicated that this isolate could be a member of a new yeast species. Three atypical Malassezia isolates M47, M54 and M235 were identified as non-lipid-dependent variants of Malassezia furfur. It was shown that in atopic dermatitis, cutaneous colonization with yeast is two-fold higher in adults than in children. The sera of atopic dermatitis patients have specific IgE antibodies to cross-reactive intracellular yeast antigens. Candida pelliculosa and house dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae might share some allergenic epitopes. The results of this study suggest that attention should be given to a cutaneous colonization by saprophytic yeast since the immune response to the allergens could further exacerbate allergic inflammation due to cross-reactive epitopes. / Išskirtos ir identifikuotos atopinio dermatito pažeistą odą kolonizuojančios mielių rūšys, įvertinta jų įtaka specifinių IgE antikūnų sintezei bei kryžminių reakcijų tarp skirtingų mielių rūšių galimybė. Nustatyta, kad 36,9 % atvejų atopinio dermatito pažeista oda yra kolonizuojama Candida, Malassezia ir Rhodotorula genties mielėmis. Išskirtas netipinėmis fiziologinėmis savybėmis pasižymintis Malassezia restricta kamienas M8 gali būti naujos rūšies atstovas. Išskirti netipinėmis fiziologinėmis savybėmis pasižymintys Malassezia genties kamienai M47, M54 ir M235 identifikuoti kaip nuo išorinio lipidų šaltinio nepriklausantys Malassezia furfur. Įrodyta, kad mielės suaugusių asmenų atopinio dermatito pažeistą odą kolonizuoja du kartus dažniau negu vaikų. Įrodyta, kad atopiniu dermatitu sergančių asmenų kraujo serume aptinkama prieš kryžmiškai reaguojančius mielių viduląstelinius antigenus nukreiptų specifinių IgE antikūnų. Taip pat nustatyta, kad Candida pelliculosa ir namų dulkių erkių Dermatophagoides pteronyssinus ir Dermatophagoides farinae alergenai gali turėti panašius epitopus. Darbo rezultatai patikimai rodo, kad atopinio dermatito pažeistą odą kolonizuojančios komensalinės mielės gali pasunkinti atopinio dermatito eigą dėl kryžmiškai reaguojančių epitopų tarp skirtingų biologinių rūšių antigenų.

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Mémoire (M.Med.Exp.)--Université du Québec à Chicoutimi, 2000. / Document électronique également accessible en format PDF. CaQCU

La dermatite atopique est une dermatose inflammatoire apparaissant préférentiellement avant l’âge de cinq ans, et évoluant naturellement vers la guérison après l’adolescence dans plus de 90% des cas. L’origine de cette amélioration spontanée n’est pas clairement établie dans cette dermatose multifactorielle dont l’étiopathogénie reste encore à déterminer (maturation du système immunitaire ?). Les liens de cette pathologie avec le psychisme sont certains : retentissement psychologique important avec altération de la qualité de vie, aggravation voire déclenchement des poussées par le stress, amélioration des lésions par psychothérapie. L’observation de la nature de la structure d’attachement est importante dans le domaine de la psychopathologie. Elle est encore peu réalisée dans le domaine da la pathologie organique. Nous avons étudié la structure d’attachement de 80 adultes ayant présenté dans l’enfance une dermatite atopique, 40 d’entre eux ayant une guérison de celle-ci depuis l’adolescence, 40 autres présentant une pérennisation des crises d’eczéma. L’évaluation de l’attachement a été réalisée avec un autoquestionnaire (CAMIR de PIERREHUMBERT) et la cotation de l’entretien d’attachement avec la méthode Q-SORT selon KOBAK. Nous avons également mesuré les symptômes psychopathologiques associés avec l’échelle SCL90-R, le niveau d’anxiété avec l’inventaire trait-état de SPIELBERGER (STAIY) et le coping avec le WCC-R de VITALIANO. Les IgE totales ont été dosées. Les sujets avec dermatite atopique ayant débutée avant trois ans ne sont pas plus insécures que ceux avec un début plus tardif de leur dermatose. Ceci fait évoquer la possibilité d’une absence de retentissement sur la structure d’attachement d’une dermatite atopique apparaissant dans les premiéres années de vie, contrairement à ce qui est noté dans toute pathologie chronique d’apparition précoce. Il est alors supposé un effet positif des soins cutanés sur la nature de la relation de la dyade mère-enfant. Par contre, le retentissement ultérieur de la dermatite atopique chez l’adulte est important, avec augmentation d’une insécurité préoccupée en termes de représentations d’attachement, une augmentation des scores de toutes les dimensions psychopathologiques mesurées et une recherche anxieuse de soutien à l’extérieur. Ce retentissement est d’autant plus important que les IgE sont élevées, ceci n’étant pas uniquement le reflet de la gravité de la maladie. Les adultes présentant une dermatite atopique pérennisée sont structurellement plus insécures que ceux qui ont guéris : si nous considèrons l’aspect stable dans le temps de la structure d’attachement, ces résultats positionneraient celle ci en tant que possible facteur dispositionnel prédictif dans l’évolution de la dermatite atopique. Le score de sécurité des patients guéris est d’autant plus élevé que les IgE sont normales. Un phénomène d’interaction entre le système d’attachement et le système adaptatif au stress, à dépendance développementale commune sur certains aspects, est évoqué. Le rôle des IgE reste à déterminer. En termes de psychologie de la santé, la structure d’attachement pourrait être considérée comme un facteur dispositionnel susceptible d’influer l’évolution de la dermatite atopique. / Atopic dermatitis is an inflammatory skin disorder which usually appears before the age of five, and heals naturally little by little after adolescence in over 90% of cases. Why this spontaneous improvement occurs is not clearly established for this multi-factor skin disorder, for which the etiopathology has yet to be determined (immune system maturity?).There is a clear relationship between this pathology and the psyche: major psychological repercussions affecting the quality of life, aggravation or triggering of skin reactions due to stress, alleviation of lesions using psychotherapy. Observation of patterns of attachment is important in the field of psychopathology, but is not often undertaken in the domain of organic pathology. We studied the patterns of attachment for 80 adults having suffered from atopic dermatitis during childhood, 40 of them had been cured since adolescence, the other 40 continued to suffer from outbreaks of eczema. Attachment was evaluated using a self-completion questionnaire (CAMIR by PIERRE HUMBERT) and the attachment interview was graded using the Q-SORT method, by KOBAK. We also measured the associated psychopathological symptoms using the SCL90-R scale, level of anxiety using SPIELBERGER’s state-trait-anxiety inventory (STAIY), and coping measurement using the WCC-R scale devised by VITALIANO. Total IgE was measured. Patients suffering from atopic dermatitis that began before the age of 3 years are not more insecure than patients whose skin disorder started later in life. This meant that there were possibly no repercussions on the pattern of attachment for cases of atopic dermatitis that had appeared in the first years of life, contrary to all reports regarding a chronic pathology appearing early in life. This implies that caring for the skin has a positive effect on the mother-child relationship. On the other hand, adults suffer from major repercussions of atopic dermatitis later on in life: increased sense of preoccupied insecurity in terms of representations of attachment, increased scores in all the psychopathological dimensions measured, and an anxious need for outside support. These repercussions are even more serious when IgE is high, but this is not solely connected with the gravity of the illness. Adults suffering from persistent atopic dermatitis are basically more insecure than those who are cured: if long-term stability in the pattern of attachment is considered, this study could place pattern of attachment as a possible predictive disposition factor in the evolution of atopic dermatitis. The security score of cured patients is all the greater when IgE is normal. The existence of an interaction between the attachment pattern and the system for adapting to stress has been suggested, since they are both dependent on development. The role played by IgE remains to be determined. In terms of health psychology, the pattern of attachment could be considered as a disposition factor likely to influence the evolution of a skin disorder.

The thesis deals with atopic eczema in children and its health and social impact. The work is divided into a theoretical and a practical part. The theoretical part is focused on explaining the basic concepts in dermatology, presenting the anatomy and physiology of skin and its specific qualities in childhood, defining the atopic eczema and outlining its etiopathogenesis, clinical presentation, treatment and nursing care as well as health and social impacts of eczema. The practical part of the thesis set up three goals. The first objective was to determine the impact of the disease on children and their families. The second objective of the research was to determine the role of a nurse in the treatment of children with atopic eczema. The third objective was to create an educational brochure for parents of children with atopic eczema. To achieve these objectives, a qualitative research based on two semistructured interviews was carried out. The first of the two interviews was conducted with mothers of sick children. The research group consisted of ten mothers, five from Prague and five from České Budějovice. The second interview was performed with nurses in GP surgeries for children and adolescents and in surgeries of dermatologists and allergists. The second research group consisted of eight nurses, four of which worked in GP surgeries for children and adolescents, two with dermatologists and two with allergists. Nurses, as well as mothers, came from Prague and České Budějovice. Attention was paid to problems that mothers and children find the most annoying. Small children under four years of age are most bothered by the itching and pain; part of these children also dislike daily skin care. Children from five years up are upset about insufficient contact with their peers and limitations in performing their activities of interest. They have to make sure they do not swear while enjoying their hobbies and their skin must not come into contact with irritants. Their mothers suffer from the unpredictability of the disease and from not knowing whether the eczema will ever disappear. They also fear that the eczema might get worse. The medical complications related to atopic eczema include Staphylococcus aureus, Candida albicans, warts, Molluscum contagiosum and herpes in eczema-affected areas. Other complications include itching, pain, sleep disorders in the mother and child, subsequent fatigue, impaired performance at school and/or work. Generally, children and mothers have positive relationships with others, yet sometimes the have to face curious looks. Mothers tend to spend less time with their partners than they would desire. As for nurses, it was found out that the primary role in patients? education is played by doctors, whereas nurses, in addition to performing their administrative duties, provide additional information and educational materials and respond to parents? practical questions. The research results may serve mainly to parents and their children with atopic eczema as a source of information about the problems they might encounter, helping them to address such problems. The output of the thesis will be an educational brochure for parents of children with atopic eczema.

Atopic dermatitis (AD) is a common, chronic relapsing inflammatory skin disease associated with cutaneous hyper-reactivity to environmental triggers that are innocuous to normal nonatopic individuals. AD affects 10% to 15% of children and 2% to 10% of adults in industrialized countries. There has been increasing interest in this disease triggered by its increasing prevalence in western societies and its contribution to the increasing health care costs. Yet, the underlying pathophysiologic and genetic mechanisms leading to the manifestation of AD are not clear. AD results from a complex interplay between environmental triggers, susceptibility genes including mutations in the keratinocyte protein filaggrin and altered immune responses resulting in allergic CD4+ T cell (Th2) immunity to epidermally encountered antigens. Regulatory T cells (Tregs) play an important role in controlling responsiveness to self-antigens and preventing autoimmune diseases, as well as in limiting inflammatory responses during inflammation and infection. Currently, studies investigating the number and function of Tregs in patients with AD have shown controversial results. It has been long established that symptoms of AD improve on exposure to sunlight. Narrowband UVB (NB-UVB) phototherapy is a common treatment modality for a variety of skin diseases. Considering the adverse effects for systemic treatment for severe adult AD, phototherapy, especially NB-UVB phototherapy may be a more practical long-term treatment. However, approximately 50% of patients over an 8-week treatment course do not improve after NB-UVB phototherapy. Therefore, it is important to identify characteristics of AD patients to determine whether they will respond to phototherapy and to avoid adverse effects for unresponsive patients. UVB exposure has also been associated with induction of Tregs in mice and increasing their numbers and/or functional capacity may offer benefit to patients with chronic AD. Active vitamin D (1,25(OH)2D3), one of the factors induced by UV-B radiation induces Tregs and is suggested to contribute to the suppressive effect of NB-UVB phototherapy. However, UV radiation could also have beneficial effects through other pathways known to affect immunoregulation. UVB exposure upregulates production of nitric oxide (NO) in the skin which also affects immune cell function. The protein filaggrin is broken down in differentiating keratinocytes to form the natural moisturizer of the skin. The gene encoding filaggrin (FLG) has been shown to be a major predisposing factor for AD. A key breakdown product is urocanic acid (UCA) which also acts as a natural sunscreen and undergoes trans-cis isomerisation on exposure to UV-B. Cis-UCA is known to modulate immune responses, however, the mechanisms of its action remain elusive. The production of all three compounds, vitamin D, cis-UCA and NO might all increase in the circulation of patients undergoing UVB phototherapy. While the immunomodulatory effect of Vitamin D is well described, cis-UCA and NO may also affect the behaviour of T lymphocytes systemically. Therefore, I investigated the effect of NO and cis-UCA on the phenotype and function of CD4+T cells and monocyte-derived dendritic cells (Mo-DCs) derived from peripheral blood mononuclear cells (PBMCs) from healthy volunteers. I also investigated the correlation between plasma concentration of 25(OH) vitamin D and nitrate, FLG genotype, circulating Tregs and clinical efficacy of NB-UVB phototherapy. My results showed that NO did not affect the phenotype of human mo-DCs and directly affected peripheral CD4+ T cells by inducing functional CD25+Foxp3+CD127-Tregs from CD4+CD25lo/- effector T cells. Moreover, NO increased expression of the of skin homing marker CLA on these Tregs, suggesting an increased ability of NO-induced Tregs to migrate to the skin. These NO-induced CD25+Foxp3+CD127-Tregs had immunosuppressive functions and inhibited autologous CD4+ T cell proliferation. Cytokines, at least IL-10, secreted by NO-treated CD4+ T cells were not sufficient for the suppressive function of NOinduced Foxp3+Tregs. The immune regulatory function of NO-induced Fopx3+Tregs required cell-cell contact and was mediated by membrane bound TGFβ and PD-1/PD-L1 but not CTLA-4. Results also showed that cis-UCA might have both pro- and anti-inflammatory effects. Cis- UCA significantly decreased the proportion of CD25hi Foxp3+ cells from activated CD4+ T cells. It also decreased the expression of vitamin D receptor in CD4+ T cells which may interfere with the immune regulatory function of vitamin D. These results suggested that there might be a fine balance between UV-induced anti-inflammatory molecules’ effect on CD4+ T cells. However, Cis-UCA also modulated CD4+ T cell directly by decreasing CD4+ T cell proliferation, decreasing phosphorylation of ERK after TCR activation, enhancing immune suppressive cytokines secretion, and inhibiting the percentage of CLA+CD4+T cells suggesting a decreased ability to migrate to the skin, . Cis-UCA also affected the phenotype and function of antigen presenting cells by decreasing the expression of HLA-DR, CD86 and CD40 on immature mo-DCs, which led to increased proportion of CD25+Foxp3+CD127- T cells when co-cultured with allogenic CD4+ T cells. Results generated from the clinical study in which all 29 patients got better after phototherapy suggested although circulating 25 (OH) vitamin D concentration was significantly increased after NB-UVB phototherapy, the change of circulating 25 (OH) vitamin D concentration did not correlate with disease improvement. This suggests that vitamin D is not the only pathway involved and that other molecules contribute to UVB-induced immune-regulation. The data also show that of the levels of circulating nitrate and the FLG genotype did not correlate with improvement / change with phototherapy. However, the expression of CD69 and CLA on circulating CD4+ T cells was decreased after treatment suggesting that UVB affected T cell activation and migration to the skin, and their importance in determining clinical responses requires further investigation. Taken together, the results from my study provide evidence that vitamin D is not the only molecule responsible for the beneficial effect of NB-UVB phototherapy. NO and cis-UCA may down-regulate immune responses by affecting human peripheral CD4+ T cells and mo- DCs phenotype and function. A further understanding of the effect of NO and cis-UCA on skin resident immune cells will provide more insights for narrowing NB-UVB phototherapy which will help to select patients that most likely to benefit from a mechanism-based treatment.