Abstract TGF-βs are cytokines that signal through the receptor complex of type I and type II receptors. Hemidesmosome (BP180, BP230, plectin/HD1, α6β4 integrin), anchoring filaments (laminin 5), and anchoring fibrils (collagen VII) form a hemidesmosomal adhesion complex that provides stable adherence of keratinocytes to the epidermal basement membrane. Nidogen, collagen IV, and laminins are components of the basement membrane, integrins are cell adhesion molecules, and tenascin-C is a matrix protein. The expression of TGF-β receptors I and II was studied in normal epidermis and lesional and non-lesional psoriatic epidermis by immunohistochemistry. TGF-β1 and TGF-β2 in suction blister fluid and serum were determined by enzyme-linked immunoassay. Suction blister fluid and serum samples were obtained from acne patients before and after oral isotretinoin treatment. Suction blister fluid samples were also obtained from healthy volunteers in two age groups from a control site and a betamethasone-pretreated site. The expression of BP180, BP230, plectin/HD1, α6 integrin, β4 integrin, laminin 5, collagen VII, collagen IV, nidogen, laminin α3 chain, and laminin β1g1 chains was studied in uninvolved dermatitis herpetiformis skin by the immunofluorescence technique. The ultrastructure of the hemidesmosomal inner plaque was studied in uninvolved dermatitis herpetiformis skin by electron microscopy. The suction blister method was used to study intact blisters, open wounds (=blister roofs removed right after blister induction) and calcipotriol-pretreated open wounds in healthy volunteers. The reepithelialization rate and the expression of BP180, BP230, plectin/HD1, β4 integrin, laminin 5, collagen VII, laminin α5 chain, laminin β1 chain, tenascin-C, αvβ5 integrin, β5 integrin, α5 integrin, and α9 integrin during reepithelialization were studied by haematoxylin and eosin stainings and the immunofluorescence technique. BP180, BP230, and plectin/HD1 expression were analyzed by body site to exclude regional variation. In normal epidermis, TGF-β receptors I and II were detected in the basal epidermis. Diffusion calculations suggest that circulation is likely to be a major source of TGF-β for TGF-β receptors in the basal epidermis. Downregulation of TGF-β receptors I and II was seen in lesional psoriatic epidermis, suggesting that hyperproliferating lesional epidermis may have lost TGF-β-mediated growth inhibition. Isotretinoin did not affect the serum TGF-β1 or TGF-β2 levels, but caused a 19% local increase in suction blister fluid TGF-β1. Betamethasone caused a 17% decrease in suction blister fluid TGF-β1, presumably due to glucocorticoid-induced vasoconstriction. Modulation of the interstitial fluid TGF-β1 concentration may be one mechanism by which isotretinoin and betamethasone mediate their effects in skin. Immunoreactivity for BP230 and plectin/HD1 was decreased in the basement membrane zone in uninvolved dermatitis herpetiformis skin in a significant proportion of the patients, suggesting distinct molecular changes in BP230 and plectin/HD1. This may be a factor contributing to blister formation. Reepithelialization rate was considerably slower in intact blisters than in open wounds and was not affected by calcipotriol. BP230 and plectin/HD1 appeared earlier in intact blisters than in open wounds. Reepithelialization took place on a continuous laminin sheath in intact blisters, but the laminin sheath in open wounds was partially discontinuous. It was a novel finding that integrin αvβ5 and integrin β5 antibodies showed divergent distributions in regenerating epidermis. The present results suggest that, in some bullous diseases, removal of the blister roof could accelerate blister healing, calcipotriol treatment does not delay wound epithelialization, a continuous laminin sheath may inhibit reepithelialization, and the formation of the hemidesmosomal inner plaque at the leading edge takes place earlier in the more slowly reepithelializing intact blisters than in open wounds.

Abstract It is a generally accepted dogma that sepsis disturbs skin function and wound healing, but surprisingly there is only remote pathophysiological evidence available behind that presumption. As the skin is the largest defensive barrier, the skin dysfunction in severe sepsis deserves more attention. In this study, the suction blister model was used to create experimental wounds. The study population included 44 patients with severe sepsis and 15 controls. The blister fluid was collected to analyse cytokine profile of the skin. The transepidermal water loss and blood flow from the wound were measured. A 4mm biopsy was taken under local anaesthesia on the first and the eighth day of the study from the healthy looking skin. Then 15 healing suction blisters were excised. Serum samples were also collected on the first day of the study. The barrier restoration was diminished, and the inflammation in the wound was more intense in severe sepsis than in the controls. The expression of the basement membrane components Laminin-332 and type IV collagen decreased during the septic disease, but increased over the next 3 months without achieving the level oft he controls. The expression of tight junction proteins remained nearly intact in the healing wound in severe sepsis compared to the controls. The expression of occludin on the leading edge of the migrating keratinocytes was more restricted and late in severe sepsis compared to the controls. The levels of the tumour necrosis factor (TNF), interleukin-10 (IL-10) and IL-6 in skin blister fluid were higher in the sepsis compared to controls. The blister fluid and serum cytokine response in the sepsis differed since the levels of epidermal growth factor, vascular endothelial growth factor, TNF and basic fibroblastic growth factor (bFGF) in the blister fluid did not correlate with the levels of serum. The septic patients with multiple organ failure had higher levels of several cytokines than patients without organ failure. Survivors had lower levels of IL-10 and bFGF in blister fluid than the non-survivors. This study offers novel findings for skin and wound healing in sepsis. Together, all the findings suggest that skin dysfunction in severe sepsis exists even when the most profound structures remain intact. Understanding these mechanisms of impaired wound healing can improve future treatments, such as the timing of surgery. / Tiivistelmä Sepsiksen ajatellaan heikentävän haavanparanemista, mutta tieteellistä näyttöä on niukasti. Iholla on keskeinen osa elimistön puolustuksessa ja tasapainon ylläpidossa, joten sen toiminnan häiriintyminen systeemisessä tulehduksessa ansaitsee suuremman huomion. Imurakkulahaavat tehtiin 44 septiselle potilaalle ja 15 kontrollille. Haavoista mitattiin veden haihtumista ja veren virtausta sekä otettiin imurakkulaneste näytteeksi sytokiinimäärityksiä varten. Tutkimuksen ensimmäisenä ja kahdeksantena päivänä otettiin 4mm biopsiat terveeltä iholta ja 15 potilaalta poistettiin näytteeksi paraneva imurakkulahaava. Seeruminäytteet otettiin tutkimuksen ensimmäisenä päivänä. Veden haihtuminen haavalta oli voimakkaampaa eli ihon barrierin palautuminen oli hidastunut septisillä potilailla verrattuna kontrolleihin. Haavassa havaittu tulehdus oli sepsiksessä voimakkaampi. Tyvikalvon komponenttien Laminiini-332:n ja tyypin IV kollageenin ilmentyminen oli vähäisempää sepsiksen aikana ja lisääntyi 3kk kohdalla, mutta ei kuitenkaan saavuttanut kontrollien tasoa. Tiivisliitosproteiinien ilmentyminen oli lähes muuttumatonta sepsiksessä kontrolleihin verrattuna. Okludiinin ilmentyminen sen sijaan paranevassa haavassa vaeltavien keratinosyyttien etureunassa oli rajoittuneempaa ja myöhäisempää sepsiksessä kuin kontrolleilla. Sytokiineistä tuumorinekroositekijä (TNF), interleukiini-10 (IL-10) ja IL-6 olivat koholla imurakkulanesteessä verrattuna kontrolleihin. Epidermaalinen kasvutekijä, verisuonten endoteelikasvutekijä, TNF ja perusfibroplastinen kasvutekijä (bFGF) pitoisuudet rakkulanesteessä erosivat seerumin pitoisuuksista eli ihon sytokiiniprofiili erosi systeemisestä sytokiiniprofiilista. Potilailla, joilla oli monielinvaurio, todettiin korkeampia sytokiinipitoisuuksia. Potilailla, jotka menehtyivät 30 vrk kuluessa, oli korkeammat pitoisuudet IL-10 ja bFGF rakkulanesteessä. Tämä tutkimus tarjoaa uutta tietoa ihosta ja haavanparanemisesta sepiksessä. Tulosten perusteella voidaan todeta, että ihon toimintahäiriö on sepsiksessä todellinen, vaikka kaikkein perustavimmat rakenteet säilyvät muuttumattomina. Toimintahäiriön mekanismien ymmärtäminen voisi auttaa septisen potilaan hoidossa, kuten kirurgisten toimenpiteiden ajoittamisessa paranemisen kannalta mahdollisimman otolliseen aikaan.