<p>Positive patch test reactions to gold are commonly seen in dermatology clinics, but it is veryunusual for the patients to actually have any clinical symptoms. It is also common with irritantreactions that are not linked to adaptive immunity. Therefore, a deeper understanding of themechanisms underlying allergic contact dermatitis (ACD) reaction, and the search for acomplementing diagnostic tool, is important.</p><p>In paper I we included three subject groups; one with morphologically positive patch testreactions to gold sodium thiosulphate (GSTS, the gold salt used in patch testing), one withnegative patch tests, and one with irritant reactions to gold. Blood samples were collected andexamined regarding the proliferation rate and which cytokines were secreted after culturingwith GSTS. We saw that the cultured lymphocytes from the allergic donors proliferated at asignificantly higher rate than the two other subject groups, and that the cells secreted cytokinesof both Th1 (Interferon (IFN) -g and Interleukin (IL) -2) and Th2 (IL-13 and IL-10) types. Theallergic donors secreted significantly higher levels of IFN-g, IL-2 and IL-13 than the two othersubject groups. Both the negative and irritant subject groups showed suppressed levels of thecytokines as compared with the unstimulated cultures, demonstrating the immunosuppressingeffects of gold.</p><p>We also examined whether any of the analyzed markers, alone or combined, could be usedas an aid for diagnosing ACD to gold. We found that the IFN-g assay yielded the highestsensitivity (81.8 %) and specificity (82.1 %), and also identified 87.5 % of the irritant group asnon-allergic.</p><p>In paper II we decided to investigate what cell types and subsets that reacted to the goldstimulation. We analyzed proliferation rate and expression of CD45RA, CD45R0, cutaneouslymphocyte-associated antigen (CLA) and the chemokine receptors CXCR3, CCR4 andCCR10. Similar to what has previously been published about nickel (Ni) allergy, the cells fromthe gold-allergic subjects that reacted to the GSTS stimulation expressedCD3+CD4+CD45R0+CLA+. However, contrary to findings in studies on Ni-reactive cells, wesaw no differences between allergic and non-allergic subjects regarding any of the chemokine receptors studied.</p><p>In conclusion, we found that analysis of IFN-g might be a useful complement to patchtesting, possibly of interest in avoiding the need for repeated tests to rule out irritant reactions.We also saw that the cells that proliferated in response to gold were memory T-cells expressingCD4 and CLA, the marker for skin-homing. However, these cells did not express elevatedlevels of any of the chemokine receptors analyzed, showing that there are both similarities anddifferences between the mechanisms for Ni allergy and gold allergy.</p>

Positive patch test reactions to gold are commonly seen in dermatology clinics, but it is veryunusual for the patients to actually have any clinical symptoms. It is also common with irritantreactions that are not linked to adaptive immunity. Therefore, a deeper understanding of themechanisms underlying allergic contact dermatitis (ACD) reaction, and the search for acomplementing diagnostic tool, is important. In paper I we included three subject groups; one with morphologically positive patch testreactions to gold sodium thiosulphate (GSTS, the gold salt used in patch testing), one withnegative patch tests, and one with irritant reactions to gold. Blood samples were collected andexamined regarding the proliferation rate and which cytokines were secreted after culturingwith GSTS. We saw that the cultured lymphocytes from the allergic donors proliferated at asignificantly higher rate than the two other subject groups, and that the cells secreted cytokinesof both Th1 (Interferon (IFN) -g and Interleukin (IL) -2) and Th2 (IL-13 and IL-10) types. Theallergic donors secreted significantly higher levels of IFN-g, IL-2 and IL-13 than the two othersubject groups. Both the negative and irritant subject groups showed suppressed levels of thecytokines as compared with the unstimulated cultures, demonstrating the immunosuppressingeffects of gold. We also examined whether any of the analyzed markers, alone or combined, could be usedas an aid for diagnosing ACD to gold. We found that the IFN-g assay yielded the highestsensitivity (81.8 %) and specificity (82.1 %), and also identified 87.5 % of the irritant group asnon-allergic. In paper II we decided to investigate what cell types and subsets that reacted to the goldstimulation. We analyzed proliferation rate and expression of CD45RA, CD45R0, cutaneouslymphocyte-associated antigen (CLA) and the chemokine receptors CXCR3, CCR4 andCCR10. Similar to what has previously been published about nickel (Ni) allergy, the cells fromthe gold-allergic subjects that reacted to the GSTS stimulation expressedCD3+CD4+CD45R0+CLA+. However, contrary to findings in studies on Ni-reactive cells, wesaw no differences between allergic and non-allergic subjects regarding any of the chemokine receptors studied. In conclusion, we found that analysis of IFN-g might be a useful complement to patchtesting, possibly of interest in avoiding the need for repeated tests to rule out irritant reactions.We also saw that the cells that proliferated in response to gold were memory T-cells expressingCD4 and CLA, the marker for skin-homing. However, these cells did not express elevatedlevels of any of the chemokine receptors analyzed, showing that there are both similarities anddifferences between the mechanisms for Ni allergy and gold allergy.

Human skin forms a biologically active barrier and maintains vital protective functions through continuous regeneration of cells within its outermost layer, the epidermis. In healthy skin, renewal of epithelial cells is a tightly regulated process in which the epidermal growth factor receptor (EGFR or HER1) and its various ligands are involved. The biological role of other EGFR family members (HER2–4) in normal and diseased human skin has gained less interest. The purpose of this work was to investigate the expression and contribution of different HERs in cultured epidermis and psoriatic skin. Epidermal regeneration was studied by fluorescence imaging of a skin explant model exposed to anti-psoriatic drugs, HER ligands or HER-blocking molecules. EGFR, HER2 and HER3 were all markedly expressed with an in vivo-like immunostaining pattern in cultured neoepidermis, whereas only low amounts of HER4 were detected at protein and mRNA levels. Re-epithelialization was associated with receptor activation. Application of HER-selective tyrosine kinase inhibitors and monoclonal antibodies reduced the proliferative activity, receptor phosphorylation and radial outgrowth from normal skin explants. Similar anti-dynamic effects were obtained with HER kinase inhibition of neoepidermis generated from psoriatic skin. Among the HER receptors, EGFR seemed to be the dominant subtype during epithelialization in vitro although HER2 and HER3 were also involved. HER2 probably functioned as a co-receptor for the kinase-deficient HER3 in neoepidermis. In vivo, expression of HER4 mRNA was detected in normal and uninvolved psoriatic skin but was virtually absent in lesional skin, a potentially important finding for HER signalling in psoriasis. This thesis demonstrates the utility of combined dynamic and biochemical analyses of re-epithelialization and highlights the role of EGFR and other HERs for epidermal growth. It also underscores the potential of HER-directed inhibition to control hyperproliferative states of the epidermis.

Diss. (sammanfattning) Umeå : Umeå universitet, 2006. / Härtill 5 uppsatser.

Cutaneous malignant melanoma (CMM) is one of the most rapidly increasing cancers in the Swedish population. The aetiology of melanoma is a complex interplay between genetics, host characteristics and environmental factors. The host characteristic with the strongest association with CMM is a phenotype with high numbers of common naevi and with dysplastic naevi. The principal environmental factor is sun exposure. Melanoma risk assessment (paper I) In a multi-national study including 986 subjects from Sweden, Denmark, the UK, Germany and the Netherlands, the ability of primary care physicians and nurses to identify individuals at increased melanoma risk was assessed. The atypical mole syndrome (AMS) scoring system for melanoma risk was used. The AMS scoring system consists of a five point check list incorporating total body naevus counts, clinically dysplastic naevi and body distribution of naevi. After brief training, the overall agreement in diagnosis between the trained personnel and experienced dermatologists was 94.5% (kappa value 0.70, p&lt;0.05). The study showed that the scoring system successfully can be taught to personnel in primary care. The naevus phenotype in a population in northern Sweden (paper II) The naevus phenotype was investigated in a population living in the inland of northern Sweden with a low melanoma incidence. Two hundred and one participants from the community of Storuman were included. The median naevus count was15 common naevi/individual, and the prevalence of dysplastic naevi was 11%. The median naevus count and prevalence of dysplastic naevi were significantly lower than previously described in populations with higher melanoma incidence and higher ambient ultraviolet exposure in southern Sweden. This geographical variation in naevus phenotype might be explained by differences in levels of sun exposure and in genotype. Cutaneous malignant melanoma in children and adolescents (papers III–V) During the years 1973 to 2002, 250 cases of primary CMM in individuals aged 0-19 years were reported to the Swedish Cancer Registry. Histological material was available for review in 87% of the cases registered during the two first decades (1973–1992). The diagnostic accuracy in the reviewed material was 88%. The melanoma incidence doubled in teenagers between the first decade (1973–1982) and the second (1983–1992). During the third decade (1993–2002) the increasing trend was broken. A decrease in incidence was noted in boys during 1993–1997, and in girls during 1998–2002. In younger children the incidence remained extremely low, only 4 cases in children aged 0–9 years were reported during the studied 30-year period. The trunk was the most common melanoma site in boys, and legs and trunk were the most common sites in girls. Superficial spreading melanoma was the most frequent subtype, followed by nodular melanoma. During the two first decades (1973–1992), the median melanoma thickness decreased from 1.5 to 0.9 mm. The melanoma-specific 5-year survival rate was 93%. The most important prognostic factor was melanoma thickness. The prognosis for thin lesions was excellent, during a median follow up time of 12 years, no tumour less than 0.8 mm was lethal according to the Registry. The results indicate that CMM in teenagers has many features in common with adult onset melanoma. The tudy also underlines the importance of not neglecting lesions suspected for malignant change in children and adolescents, as early detection and removal is crucial for the prognosis also in this young age group. / The electronic version of the thesis is a corrected version of the printed version.

Keloids are a fibroproliferative disorder of unknown etiology developing in the skin after injury or spontaneously. The aim of this thesis is to gain deeper insight into the role of TGF-β and its signaling pathway proteins, SMADs, in the pathogenesis of keloids and describe the gene expression profile in different keloid sites in the search for potential target genes for future treatment. Further aim is to develop an instrument to describe the quality of life of patients with keloids. We find cultured keloid fibroblasts to express an increased level of TGF-β1 mRNA and a decreased level of TGF-β3 mRNA compared to control skin. Keloid derived fibroblasts exhibit significantly decreased mRNA levels of TGF-β receptor type II (TβRII) and the ratio of TβRI/TβRII mRNA expression is increased. This suggests that a certain expression pattern of TGF-β subtypes and receptors may be important in keloid pathogenesis. Analysis of keloid derived fibroblasts reveal decreased SMAD3 mRNA expression and decreased ratio of SMAD2/SMAD3 mRNA implicating a disturbed SMAD signaling. Keloid fibroblasts up-regulate SMAD4 protein after stimulation with TGF-β1 and display diminished levels of the inhibitory proteins SMAD6 and 7. This may contribute to unlimited and deregulated TGF-β signaling leading to increased extracellular matrix production (ECM). The gene expression pattern is described in fibroblasts from different keloid sites using microarrays covering the whole human genome. This study reveals 105 regulated genes (79 genes are up- and 26 down-regulated) resulting in a unique gene expression profile in different sites of keloids, where progression or regression of the keloid process took place. In cells from the central part of keloids with clinical signs of regression, an up-regulation of apoptosis inducing genes as ADAM12 and ECM degrading genes as MMP19 is found. These genes may contribute to regression of keloids and might be possible future target genes for treatment. Overexpression of apoptosis inhibitors as AVEN and down-regulation of angiogenesis inhibiting genes as PTX3 found at the active margin of keloids may be responsible for the invasive character of the keloid margin. We develop a disease specific questionnaire to measure the quality of life of patients with keloids. We find two scales, psychological and physical impairment, describing the dimensions of quality of life in patients with scars. These two scales are independent of each other and show a high test-retest reliability. Single items which clinically characterize the disease show correlations to these scales. The results of this study demonstrate for the first time a severe impairment of quality of life of patients suffering from keloids and hypertrophic scars. In conclusion the described alteration in TGF-β expression and its receptors, the disrupted SMAD signaling pathway and the unique gene expression patterns in different keloid sites provide new knowledge on ECM formation and degradation in keloids. Regulatory genes in ECM homeostasis may be future target genes for keloid prevention, regression and treatment. The disease specific quality of life instrument of patients with keloids and scars is a useful tool to estimate success in future therapeutic efforts over time.

Malignant melanoma is one of the most rapidly increasing cancers and accounts for about three-quarter of all skin cancer deaths worldwide. Despite compelling evidence that ultraviolet (UV) irradiation causes melanoma the knowledge how various wavelength spectra affect the balance between proliferation and apoptosis controlling the homeostasis of the melanocyte population is still limited. The aim of this thesis was to elucidate the regulation of UVA/B induced apoptotic signaling in human epidermal melanocytes in vitro in relation to redox alterations and antioxidant photoprotection. UVA irradiation induced changes in plasma membrane stability, decreased cell proliferation and increased apoptosis. In comparison, melanocyte plasma membrane was markedly resistant to UVB irradiation although apoptosis was triggered. Thus, UVA irradiation should not be overlooked as an etiologic factor in melanoma development. Further, after irradiation with UVA/B we found alterations in redox state manifested by a reduction of intracellular GSH levels, translocation of nuclear factor-κB from the cytosol to the nucleus, an increase of γ-glutamylcysteine synthetase, the rate-limiting enzyme in GSH synthesis, and an increased apoptosis frequency. α-Tocopherol provided photoprotection through several modes of action affecting redox alterations and signaling, stabilizing the plasma membrane, and decreased proliferation and apoptosis rate, while β-carotene did not show the same protective capacity. Altogether, α-tocopherol might be a useful substance in protecting melanocytes from UV induced damage. We demonstrate UVA/B irradiation to activate the intrinsic pathway of apoptosis in melanocytes where translocation of Bcl-2 family proteins to the mitochondria modulates the apoptosis signal. Interestingly, the anti-apoptotic Bcl-2 family proteins generally thought to be attached to membranes, were localized in the cytosol before UV irradiation and translocated to the mitochondria in the surviving population, which might be a critical event in preventing apoptotic cell death. Lysosomal cathepsins were released to the cytosol acting as pro-apoptotic mediators upstream of activation and translocation of Bax to the mitochondria. When melanocytes were exposed to UVA, p53 participated in apoptosis regulation through interaction with Bcl-2 family proteins, while UVB induced p53-transcriptional activity and apoptosis involving lysosomal membrane permeabilization. Thus, depending on the UV wavelength p53 mediated apoptosis in melanocytes by transcriptional dependent or independent activity. These results emphasize p53 as an important pro-apoptotic component in the regulation of apoptosis. This thesis gives new insight in the harmful and various effects of different wavelengths within the UV spectrum on human melanocytes in vitro. Improved knowledge of the apoptosis regulatory systems in melanocytes might lead to a better understanding of the formation of pigment nevi and malignant melanoma and, in the future, provide better strategies to prevent and eliminate tumor development and progression.

<p>Hereditary ichthyosis is a collective name for many dry and scaly skin disorders ranging in frequency from common to very rare. The main groups are autosomal recessive lamellar ichthyosis, autosomal dominant epidermolytic hyperkeratosis and ichthyosis vulgaris, and x-linked recessive ichthyosis. Anhidrosis, ectropion and keratodermia are common symptoms, especially in lamellar ichthyosis, which is often caused by mutations in the transglutaminase 1 (TGM1) gene. The aim of this work was to study patients with different types of ichthyosis regarding (i) the patho-aetiology (TGM1 and electron microscopy [EM] analysis), (ii) skin signs and symptoms (clinical score and subjective measure of disease activity), (iii) quality of life (questionnaires DLQI, SF-36 and NHP and face-to-face interviews) and (iv) a search for new ways of topical treatment. Patients from Sweden and Estonia with autosomal recessive congenital ichthyosis (n=83) had a broader clinical spectrum than anticipated, but a majority carried TGM1 mutations. Based on DNA analysis and clinical examinations the patients were classified into three groups, which could be further subdivided after EM analysis. Our studies indicate that patients with ichthyosis have reduced quality of life as reflected by DLQI and by some domains of SF-36, by NHP and the interviews. All the interviewees reported that their skin disease had affected them negatively to varying degrees during their entire lives and that the most problematic period was childhood. All patients with ichthyosis use topical therapy. In a double-blind study creams containing either 5% urea or 20% propylene glycol were found inferior to a cream formulation containing lactic acid 5% and propylene glycol 20% both regarding clinical improvement and thinning of the skin barrier. Improved topical therapy may reduce the need of more toxic, oral drugs. Future studies should elucidate whether this increases the quality of life of ichthyosis patients, especially if combined with more detailed information about the aetiology and inheritance of the diseases.</p>

Hereditary ichthyosis is a collective name for many dry and scaly skin disorders ranging in frequency from common to very rare. The main groups are autosomal recessive lamellar ichthyosis, autosomal dominant epidermolytic hyperkeratosis and ichthyosis vulgaris, and x-linked recessive ichthyosis. Anhidrosis, ectropion and keratodermia are common symptoms, especially in lamellar ichthyosis, which is often caused by mutations in the transglutaminase 1 (TGM1) gene. The aim of this work was to study patients with different types of ichthyosis regarding (i) the patho-aetiology (TGM1 and electron microscopy [EM] analysis), (ii) skin signs and symptoms (clinical score and subjective measure of disease activity), (iii) quality of life (questionnaires DLQI, SF-36 and NHP and face-to-face interviews) and (iv) a search for new ways of topical treatment. Patients from Sweden and Estonia with autosomal recessive congenital ichthyosis (n=83) had a broader clinical spectrum than anticipated, but a majority carried TGM1 mutations. Based on DNA analysis and clinical examinations the patients were classified into three groups, which could be further subdivided after EM analysis. Our studies indicate that patients with ichthyosis have reduced quality of life as reflected by DLQI and by some domains of SF-36, by NHP and the interviews. All the interviewees reported that their skin disease had affected them negatively to varying degrees during their entire lives and that the most problematic period was childhood. All patients with ichthyosis use topical therapy. In a double-blind study creams containing either 5% urea or 20% propylene glycol were found inferior to a cream formulation containing lactic acid 5% and propylene glycol 20% both regarding clinical improvement and thinning of the skin barrier. Improved topical therapy may reduce the need of more toxic, oral drugs. Future studies should elucidate whether this increases the quality of life of ichthyosis patients, especially if combined with more detailed information about the aetiology and inheritance of the diseases.

<p>Myelomatose (benmargskreft) er en blodsyk dom som rammer ca 200 nordmenn årlig. Sykdommen kan ikke kureres og karakteriseres av symptomer som benmargssvikt og infeksjonstendenns, men kanskje først og fremst av sykelig nedbrytning av skjelettet. Pasientene rammes i høy utstrekning av benbrudd, hvirvelsammenfall og skjelettsmerter. Mekanismene for bennedbrytning og vekstkontroll står sentralt i avhandlingsarbeidet som består av fem artikler om cytokiners rolle i myelomatose. Cytokiner er signalsubstanser som benyttes i celle-celle-kommunikasjon. Det er sannsynligvis ubalanse av cytokiner som forårsaker den sykelige nedbrytningen av bensubstansen. </p><p>Det første delarbeidet omhandler funnet av hepatocyttvekstfaktor (HGF) som er uttrykt hos nesten alle pasienter med myelomatose Dette påvises med forskjellige teknikker og det benyttes bl a en separasjonsmetode for myelomceller basert på Ugelstadkuler som ble utviklet ved IKM i 1993. Videre påvises forhøyede nivåer av HGF i serum fra pasienter. Et interessant funn er at HGF reseptor også er uttrykt i pasientprøver, hvilket kan tale for at myelomceller kan ha en selvstimulerende (autokrin) funksjon.</p><p>I det andre delarbeidet vises en dyremodell for myelomatose i immundefekte mus. Et hovedpoeng er at det lar seg gjøre å få vekst av myelomceller i musebenmarg med påvisbare tegn til patologisk bennedbrytning på røntgen og ved histologisk undersøkelse. Musene har forhøyede nivåer av HGF i serum. Benlesjonene ble karakterisert ved hjelp av histomorfometri. Denne undersøkelse viste 99% reduksjon av de bendannende cellene (osteoblaster) og 33% reduksjon av bennedbrytende celler (osteklaster).</p><p>I tredje delarbeidet viser man at HGF induserer interleukin (IL)-11-produksjon i osteoblaster. IL-11 er en kjent påskynder av benresorpsjon og osteoklastaktivator. Et interessant fenomen er at HGF ser ut til å være bundet til heparansulfat på cellemembranen og at slikt membranbundet HGF virker bedre enn løselig HGF. Effekten av HGF potensieres av cytokinene TGF-beta og IL-1. En styrke ved arbeidet er at såvel ferskisolerte pasientceller som cellelinjer viser identiske mønstre. Arbeidet angir en mulig måte som HGF kan befremme bennedbrytning.</p><p>I fjerde delarbeid vises at cytokinet IL-15 forhindrer programmert celledød (apoptose) i myelomcellelinjen OH-2. Det var fra før kjent at myelomceller relativt hyppig lar seg stimulere av cytokinet IL-6, som fortsatt er den mest anerkjente myelomvekstfaktoren. IL-15 var tilnærmet like potent antiapoptotisk som IL-6, og befremmet også kortvarig proliferasjon. IL-15s effekt kunne potensieres av TNF-alfa </p><p>I femte delarbeid påvises at cytokinet benmorfogent protein (BMP)-4 hemmer vekst av myelomceller. BMP-4 befremmer bendannelse. Effekten av BMP-4 kom fram i IL-6-stimulerte cellelinjer og pasientprøver. Effekten skyldtes såvel induksjon av apoptose som stopp i cellesyklus G1-fase. Dette er et mulig viktig funn siden man kan tenke seg at pasienter med myelomatose kunne behandles med BMP-4 eller lignende substanser. På slik måte ville såvel skjelettnedbrytningen som myelomcellevekst kunne påvirkes gunstig. </p><p>Arbeidet bidrar til forståelse av molekylære mekanismer for bendestruksjon og myelomcellevekst og ble veiledet av profesor dr. med. Anders Waage. Henrik Hjorth-Hansen har vært stipendiat i Den norske kreftforening, og undersøkelsen ble dessuten støttet av Kreftfondet ved RiT og Blix’ legat. </p>