Notes: Radiation-induced fibrosis is a common side-effect of cancer treatment. The pathophysiological events leading to fibrosis are not known in detail. We analysed the effect of therapeutic irradiation on human skin collagen synthesis, skin thickness, gelatinases and their inhibitors. Twenty randomly chosen women who had been treated for breast cancer with surgery and radiation therapy participated in the study. In each patient, the irradiated skin area was compared with a corresponding non-treated skin area. Suction blister fluid (SBF) and serum samples were analysed for the aminoterminal propeptides of type I and type III procollagens (PINP and PIIINP), tissue inhibitors of matrix metalloproteinases (MMPs) 1 and 2 (TIMP-1 and TIMP-2) and MMP-9 and MMP-2/TIMP-2 complex. Skin biopsies were analysed for PINP and immunohistochemical staining was used for PIIINP. In irradiated skin, PINP, PIIINP, TIMP-1 and MMP-2/TIMP-2 complex levels in SBF and the number of PINP-positive fibroblasts in tissue sections were significantly higher in comparison with non-treated skin. The levels of TIMP-2 in irradiated and non-irradiated skin were similar. MMP-9 could not be detected in SBF with the assay used. The serum levels of MMP-9 were higher in the treated subjects than the reference values. The serum values of PINP, PIIINP, TIMP-1, TIMP-2 and MMP-2/TIMP-2 complex were not significantly affected. These results indicate increased local collagen synthesis and accumulation of connective tissue in irradiated skin. The marked upregulation of collagen synthesis as a result of irradiation offers a possibility to treat this complication with compounds such as topical steroids which downregulate collagen synthesis.

Notes: The effects of topical tretinoin on collagen synthesis and degradation were studied in 29 volunteers. The subjects applied 0·1% tretinoin cream on their non-sun-exposed abdominal skin once a day for 1 week (n = 10) (experiment 1) or twice a day for 2 weeks (n = 8) (experiment 2) or once a day for 2 months (n = 11) (experiment 3). After the treatments, suction blisters were induced and aminoterminal propeptides of type I and III procollagens (PINP, PIIINP, respectively) (experiments 1 and 3) and carboxy-terminal propeptide of type I procollagen (PICP) (experiment 2) were assayed as an index of de novo collagen synthesis by radioimmunoassays. Matrix metalloproteases 2 (MMP-2) and 9 (MMP-9) were assayed by the zymography method in experiment 2. In experiment 3, histology, immunohistochemistry of type I and III procollagens, tenascin. mRNA levels of type I collagen α1-chain [α1(I)], interstitial collagenase (MMP-1). MMP-2, MMP-9 by slot-blot analysis and the levels of α1(I) collagen mRNA by a quantitative polymerase chain reaction method were studied. The proportional area of elastic fibres visualized in Verhoeff-stained sections was analysed by computerized digital image analysis. The results indicated that treatment with topical tretinoin does not markedly induce de novo synthesis of collagen in vivo or affect matrix metalloproteases. In the immunohistochemical stainings, tenascin was increased in the papillary dermis. As it has been suggested that tretinoin could counteract the atrophogenic effect of corticoids on the dermis, the effect of a combination of betamethasone-17-valerate (once a day) and tretinoin (once a day) on the propeptide levels was also studied. Betamethasone alone caused a 60% decrease in the concentrations of PINP and PIIINP, and a similar decrease was found after the combination treatment, indicating that topical tretinoin administered during short treatment periods does not counteract the inhibitory effect of a potent corticoid on collagen propeptides.

Notes: We have investigated re-epithelialization following induction of suction blisters in humans in intact blisters, open wounds, i.e. blister roofs removed immediately after blister induction, and calcipotriol-pretreated open wounds. Intact blisters simulate blister healing in bullous disease, while open wounds simulate re-epithelialization during wound healing. Re-epithelialization was clearly faster in open wounds than in intact blisters, and was not affected by calcipotriol pretreatment. Bullous pemphigoid antigen 2 (BP180), bullous pemphigoid antigen 1 (BP230), plectin/hemidesmosomal 1 protein (HD1), laminin 5, laminin α5, laminin β1, type VII collagen, tenascin-C, β4, αvβ5, α5 and α9 integrins were studied in intact blisters and open wounds by immunohistochemistry. Hemidesmosomal plaque proteins BP230 and plectin/HD1, which connect the keratin cytoskeleton to the hemidesmosome, appeared earlier at the leading edge in intact blisters than in open wounds. Band-like immunostaining in the basement membrane for laminin 5, α5 and β1 chains was continuous in blister bases, but partially discontinuous in open wound bases. The other antigens studied showed similar expression in intact blisters and open wounds. BP180, BP230, plectin/HD1, β4 integrin, laminin 5 and tenascin-C expression were further studied in calcipotriol-pretreated open wounds. Calcipotriol did not affect the expression of these antigens. The immunohistochemical results suggest that the keratin cytoskeleton is linked to the basal plasma membrane of migrating basal cells via BP230 and plectin/HD1 earlier in the more slowly re-epithelializing blisters than in open wounds. An intact laminin sheath may inhibit keratinocyte migration in intact blisters.

Notes: Laminin-5 is a glycoprotein which mediates epithelial cell adhesion to the basement membrane. This study describes the distribution and synthesis of laminin-5 in oral lichen planus, epithelial dysplasias, squamous cell carcinomas and a lymph node metastasis using immunohistochemistry and in situ hybridization. In normal oral mucosa and lichen planus, immunoreaction to the laminin-5 was seen as a thin continuous, delicate line in the basement membrane region, although slight irregularities in the thickness and intensity of the immunoreaction could be detected in some cases with lichen planus. In epithelial dysplasias, the laminin-5 staining was discontinuous and more diffuse compared to lichen planus and normal mucosa. The immunoreaction was generally extracellular, although in some cases with lichen planus and epithelial dysplasia there were a few basal epithelial cells showing cytoplasmic staining. The invasive carcinomas and the lymph node metastasis showed a striking, intense cytoplasmic, staining of the carcinoma cells along the invasive border of the neoplastic islands and in individual infiltrating carcinoma cells. Using in situ hybridization, the laminin-5 γ2 chain mRNA expression could not be detected in normal oral mucosa whereas, in non-dysplastic lichen planus and, more strongly, in dysplasias, there was a clear increase in the expression of laminin-5 mRNA in the basal epithelial cells. The most intensive signal was detected in the invasive front of the oral squamous cell carcinomas and the lymph node metastasis. We conclude that, in oral squamous cell carcinoma, there is altered synthesis and secretion of laminin-5 mRNA and protein. It is also evident that in dysplastic lesions of oral epithelium the synthesis and distribution of laminin-5 is abnormal.

Notes: Human skin is composed of epidermal and dermal layers, each of which has its own functional importance. Dermis consist of a fine network of collagen fibers, elastic fibers, and other components of the extracellular matrix (ECM). ECM consist primarily of proteins and complex sugars, which form fibrillar networks and a ground substance. Collagen is an important structural component of skin connective tissue and provides the tensile strength of skin.Approximately 70–80% of the dry weight of skin consists of collagen. The most abundant collagen types in skin are types I and III; the former accounts for 80% of the total collagen content of skin and the latter for approximately 15%. The other collagen types present in skin include type IV collagen, which is abundant in the basement membrane (BM); type V collagen, which is located pericellularly; type VI collagen, which plays a role in matrix assembly and is present as microfibrils between collagen fibers; and type VII collagen, which is a structural component of anchoring fibrils. Elastin accounts for only about 1–2% of the dry weight of skin but is important for the maintenance of skin elasticity and resilience. Glycosaminoglycans are of central importance for the maintenance of a water balance in skin, even though the quantities in ECM are small (0.1–0.3% of the dry weight of skin).In the dermis fibroblasts are responsible for the synthesis of ECM proteins. The fibroblasts in the dermis spend majority of time in quiescent state. However in response to activation, the fibroblasts can be reactivated, and certain pool of cell is able to differentiate into myofibroblasts which have important role in repairing skin defects such as during wound healing. During aging the number of fibroblasts is markedly reduced.Also the response of fibroblasts to various growth factors and mechanical or pathological stimulates (wound healing) is diminished.Skin collagen synthesis declines with aging and as the result of such external factors as long-term sun exposure and medications, for example, topical corticosteroids. In aging skin, collagen fibers become thicker and less soluble and the synthesis of collagen declines. Skin thickness remains quite constant between 20 and 70 years of age, after which a marked decrease in skin thickness occurs. During aging the expression of collagenases are increased and inhibitors of collagenases are reduced leading to increased proteolysis of connective tissue. Recent studies have shown that collagen synthesis is declined in the skin of heavy smokers, while collagenases are increased inducing premature skin aging.The elastic properties of skin are also affected by aging. Along with increasing age, dermal elastic fibers become thicker and fragmented and oxytalan fibers appear fragmented and shortened. Disintegration of elastic fibers is already seen in a minority of fibers between ages 30 and 70, but the changes become more profound after the age of 70 years, affecting a majority of the fibers. As a result of the decreased number of elastic fibers in aged skin, the elastic recovery of skin decreases in elderly people. Even though the content of GAGs and proteoglycans is relatively small, they have significant role in collagen fibril formation, water content of dermis and in mechanical properties. During aging there are marked alterations in different proteoglycans. The amount and synthesis of versican (high molecular size) is decreased and small molecular size decorin is increased. In photoaged skin versican is increased and is closely associated to elastin while decorin is decreased.

Notes: Sixty-two skin samples from patients with a variety of benign disorders (20 cases of psoriasis, 14 cases of chronic dermatitis, 11 seborrhoeic keratoses, 11 cases of lichen planus), and seven normal skin samples, were stained immunohistochemically with a polyclonal antibody (CM-1) to p53, and a monoclonal antibody to Ki67, using the avidin-biotin complex method, p53-positive keratinocytes could be found in most of these lesions. The percentage of p53-positive cells was, however, far lower than usually seen in p53-positive malignant tumours. No p53 reactivity was observed in the normal skin samples. Variable Ki67 reactivity was observed in all skin samples. Overall, the number of Ki67-positive cells was higher in skin samples in which the proportion of p53-positive cells was high (〉0.5% of total epidermal cell population) (P=0.004). This also applied separately to psoriatic and non-psoriatic lesions (P=0.028 and P=0.033, respectively). In cases with 〉10% of Ki67-positive cells, there were significantly more mitoses (P〈0.001). This association applied to both psoriasis and the other lesions studied (P=0.024 and P 〈0.001, respectively). The results show that immunohistochemically detectable accumulation of p53 is a frequent finding in non-neoplastic skin lesions. As p53 positivity was associated with the proliferation marker Ki67, the accumulation of p53 is possibly a response to an increased proliferation rate of the keratinocytes in these skin diseases, or alternatively it may be associated with apoptosis.

Notes: Summary In the present study, the recovery of the collagen synthesis rate after topical potent glucocorticoid treatment in the human skin in vivo was investigated. In the first experiment, two age groups were compared: young subjects with an age range of 21–26 years (mean 23), and old subjects, aged 55–70 years (mean 64). Twenty healthy male volunteers applied betamethasone-17-valerate to their abdominal skin for 3 days twice a day. Suction blisters were induced on the treated areas, and on the opposite side (healthy non-treated skin), of the abdominal skin on the day following the discontinuation of the treatment, and on the second and seventh day. In another experiment, suction blisters were induced after the treatment and 2 weeks later on the treated area and on healthy skin, in eight male volunteers. In both experiments, the aminoterminal propeptides of type I and III collagens (PINP and PIIINP, respectively) were measured radioimmunologically from the suction blister fluid. Corticosteroid treatment decreased the collagen synthesis in both age groups after a 3-day treatment period, and essentially no recovery in the collagen synthesis could be seen during a 1-week corticoid-free period. The inhibition and downregulation of collagen synthesis in the corticoid-treated skin was similar in both young and old subjects, up to 7 days after the treatment. During the 2-week corticoid-free period, collagen synthesis was recovered to about 50% of the level seen in the non-treated skin. Indicating that collagen synthesis is not completely normalized in the human skin even during a 2-week corticoid-free period.

Notes: Types I and III collagen are the main fibrillar collagens in the skin. Marked changes occur in the biosynthesis of these proteins during treatment with various drugs, upon ageing and in several diseases. Since conventional methods of assessing these changes have several disadvantages, a new method for estimating collagen synthesis in human skin in vivo has recently been developed. In this method suction blisters are induced on intact, and treated or diseased skin and types I and III procollagen propeptides are measured radioimmunologically in suction blister fluid (SBF).〈section xml:id="abs1-2"〉〈title type="main"〉Conclusions: The concentrations of type I and III procollagen propeptides in SBF reflect the corresponding local ongoing skin collagen synthesis. This method offers a new sensitive tool for experimental and clinical dermatology for monitoring skin collagen synthesis. With this method it has been possible, for example, for the first time directly to show in vivo that glucocorticoids rapidly and dramatically decrease human skin collagen synthesis.