Human Skin Organ Culture Research Articles

In Vitro Cutaneous Biotransformation of Propranolol

The metabolism of propranolol by human skin and by several cell preparations has been investigated in vitro. The major metabolites produced by human skin in organ culture and by keratinocytes were N-desisopropylpropranolol (DIP), propranolol glycol (GLY), and naphthoxylactic acid (NLA). Formation of GLY and NLA was linear with incubation time up to 6 d and was directly proportional to propranolol concentration. Fibroblasts and melanocytes also produced GLY and NLA, but appeared to have lower propranolol-biotransforming activity than keratinocytes. The three metabolites detected arise from side-chain oxidation of propranolol, and the use of specific enzyme inhibitors determined that monoamine oxidase and cytochrome P450 isozymes are involved in their formation. Aldehyde and alcohol dehydrogenases are also probably involved in the formation of NLA and GLY, but attempts to inhibit these enzyme systems were inconclusive, possibly due to the chemical instability of the intermediate aldehyde resulting from monoamine oxidase activity. No evidence was found for conjugation or ring oxidation by the skin or isolated cells. Induction of keratinocyte differentiation with Ca++ or phorbol ester treatment resulted in an increase of overall biotransformation and the NLA/GLY ratio.

Phenytoin has little effect on in-vitro models of wound healing

It has been reported that phenytoin induces gingival and connective tissue hyperplasia and may be of use in wound healing. In this study the effect of phenytoin on human epidermal keratinocytes and skin fibroblasts has been investigated in vitro. Cell cultures were exposed to increasing concentrations of phenytoin from 10(-9) to 10(-4) M in the presence of 1 and 10% serum supplemented medium. In addition the effect of phenytoin on epidermal cell migration (epiboly) has been investigated using organ culture of human skin. No stimulation of cell growth was observed, and only a mild toxicity affecting keratinocytes was seen at the highest concentrations. Similarly, no effect on epidermal cell migration in vitro was observed. The lack of a direct effect in vitro suggests that any in-vivo effect was not the result of interaction between phenytoin and keratinocytes or fibroblasts but possibly due to indirect modulation via other cell types, such as inflammatory or lymphoreticular cells.

Degradation of Newly Synthesized High Molecular Mass Hyaluronan in the Epidermal and Dermal Compartments of Human Skin in Organ Culture

Human whole skin was labeled for 24 h with [6-3H]-glucosamine in organ culture and epidermis, dermis and culture medium were separately analyzed for the molecular mass and content of the [3H]-labeled hyaluronan (HA). Gel filtration on Sephacryl S-1000 of HA purified by HPLC showed a large proportion of the newly synthesized HA to be of a very high molecular mass (greater than 2 X 10(6) Da) in both epidermis and dermis, whereas HA in the medium was of a smaller size. After 24 h chase, most of the high molecular mass HA, and 42-48% of total labeled HA disappeared from both tissue compartments. The size of labeled HA recovered in the chase media was further reduced but the content roughly corresponded to that lost from tissue. The amount of unlabeled HA was not significantly altered in epidermis, whereas in dermis it was reduced to about 10% of the initial values during 5-d culture. The results demonstrate that HA of both epidermis and dermis is synthesized as a very high molecular mass compound but rapidly undergoes a limited degradation into large fragments. The fragmentation of HA is suggested to enhance its diffusion from the tissues, particularly dermis.

Effects of nicotinamide on biochemical changes and microblistering induced by sulfur mustard in human skin organ cultures

Blister formation due to sulfur mustard (HD) exposure was studied in an ex vivo human skin model. Pieces of fresh human skin were exposed to HD vapor and subsequently incubated in medium for 48 hr. During this culture period cellular NAD + levels and uptake of glucose from the medium decreased relative to the duration of the exposure to HD. In light microscopic sections of skin that were exposed to HD for 6 min, clefts of variable size could be clearly observed between the epidermis and the dermis after a 48-hr culture period. Immunohistochemical staining of collagen type IV demonstrated that separation occurred between the basal membrane and the basal keratinocytes. The described ex vivo human skin model mimicked the in vivo process quite well. Since it was reported that nicotinamide could protect cellular NAD + levels after HD exposure, HD-treated skin pieces were incubated in medium containing 10 m m nicotinamide. Although this incubation caused an elevation of cellular NAD + levels and of glucose uptake from the medium compared to control values, it did not result in a substantial reduction of cell death or microblister formation as observed by light microscopy in tissue sections. It was concluded that depletion of cellular NAD + levels is not the only cause of HD-induced cell death.

Expression of Extracellular and Intracellular Bullous Pemphigoid Antigens at the Dermal-Epibolic Junction in Organ Culture of Human Skin

Recent papers have reported that there are at least two distinct binding sites of bullous pemphigoid (BP) antibodies; one is the extracellular (lamina lucida) BP antigen (E-BPA) and the other is the intracellular (basal cell cytomembrane-hemidesmosome-cytoskeleton) BP antigen (I-BPA). In attempting to characterize the properties of these two BP antigens (BPA), we have investigated the expression of E-BPA and I-BPA as well as other basement membrane zone (BMZ) components such as type IV collagen, laminin, and epidermolysis bullosa acquisita (EBA) antigen at the dermal-epibolic junction as a model system for BMZ neogenesis. I-BPA was found along the dermal-epidermal junction and throughout the whole length of the dermal-epibolic junction. However, E-BPA and other BMZ components were found along the dermal-epidermal junction and along only the most proximal portion of the dermal-epibolic junction. These results suggest 1) that emergence of E-BPA varies from that of I-BPA in such a model system of BMZ neogenesis, and 2) that the emerged E-BPA may attach to the newly formed basement membrane complex.

Formation of DNA Adducts in the Skin of Psoriasis Patients, in Human Skin in Organ Culture, and in Mouse Skin and Lung Following Topical Application of Coal-Tar and Juniper Tar

Preparations of coal-tar and juniper tar (cade oil) that are used in the treatment of psoriasis are known to contain numerous potentially carcinogenic polycyclic aromatic hydrocarbons (PAH). Evidence of covalent binding to DNA by components of these mixtures was sought in a) human skin biopsy samples from 12 psoriasis patients receiving therapy with these agents, b) human skin explants maintained in organ culture and treated topically with the tars, and c) the skin and lungs of mice treated with repeated doses of the formulations following the regimen used in the clinic. DNA was isolated from the human and mouse tissues and digested enzymically to mononucleotides. 32P-Post-labeling analysis revealed the presence of aromatic DNA adducts in the biopsy samples at levels of up to 0.4 fmol total adducts/microgram DNA. Treatment of human skin in organ culture produced similar levels of adducts, while treatment with dithranol, a non-mutagenic therapeutic agent, resulted in chromatograms indistinguishable from those from untreated controls. In mouse skin, coal-tar ointment and juniper tar gave similar DNA adduct levels, with a similar time-course of removal: maximum levels (0.5 fmol/microgram DNA) at 24 h after the final treatment declined rapidly to 0.05 fmol/microgram at 7 d, thereafter declining slowly over the succeeding 25 d. However, while coal-tar ointment produced only very low levels of adducts in mouse lung (less than 0.03 fmol/microgram DNA), juniper tar produced adducts at a high level (0.7 fmol/microgram DNA) that were persistent in this tissue. These results provide direct evidence for the formation of potentially carcinogenic DNA damage in human and mouse tissue by components of these therapeutic tar preparations.

Vitamin A Esterification in Human Epidermis: A Relation to Keratinocyte Differentiation

Keratinocytes from three different layers of epidermis (stratum basale, stratum spinosum, and stratum granulosum/corneum) were shown by high-performance liquid chromatography to contain retinol, 3,4-didehydroretinol and several fatty acyl esters thereof. The concentration of unesterified congeners increased 1.8-2.8 times from the inner to the outer layers of epidermis, while the corresponding increase in fatty acyl esters was 4.0-6.5 times. Together the esters represented 71% of the total vitamin A content in stratum granulosum/corneum as compared to 54% in stratum basale. The in situ synthesis of fatty acyl esters of retinol and 3,4-didehydroretinol (vitamin A2) was studied by addition of [3H]retinol to organ-cultured human breast skin. The radioactive compounds appearing in the epidermis after 48 h were, in order of abundance, retinyl esters, retinol, 3,4-didehydroretinyl esters, and 3,4-didehydroretinol. Studies at the subcellular level demonstrated the highest esterifying activity in the microsomal fraction. The enzyme catalyzing the reaction, acyl CoA:retinol acyltransferase (ARAT; EC 2.3.1.76), had a pH optimum of 5.5-6.0, which differs from that of ARAT in other tissues. ARAT activities in microsomes from different layers of epidermis were similar, but, owing to a presumed pH gradient in upper epidermis, the in vivo esterification of vitamin A may be enhanced in terminally differentiating keratinocytes. The mean ARAT activities in basal cell carcinomas and squamous cell carcinomas were less than 50% of the control values, and the relative amounts of retinyl esters were significantly lower than normal. We suggest that the esterification of vitamin A may also be of importance in relation to pathologic keratinocyte differentiation.

Hyaluronate Accumulation in Human Epidermis Treated with Retinoic Acid in Skin Organ Culture

Retinoic acid (RA) has been shown to retard the differentiation of epidermal keratinocytes by several morphologic and biochemical criteria. In this study, the epidermal content and localization of hyaluronate (HA), as well as its synthesis and disappearance in human skin organ culture, were characterized to test the idea that some of the RA influences on epidermal differentiation are associated with keratinocyte HA metabolism. RA stimulated the incorporation of 3H-glucosamine into HA by up to 60% at concentrations between 50 nM and 5 microM, while pulse-chase experiments revealed little change in its disappearance rate from epidermis. After 5 d in culture, the chemically quantified HA was more than doubled in the treated epidermis. The accumulation of HA was substantiated by light and electron microscopy with a specific probe prepared from the HA binding region of cartilage proteoglycan. The staining was particularly enhanced between the upper spinous cell layers, where the terminal differentiation into corneocytes normally takes place. A patchy, discontinuous staining was also seen in stratum granulosum and corneum layers, which are not stained at all in control cultures. The present study demonstrates that RA leads to an accumulation of HA in the superficial layers of epidermis by stimulating its synthesis in keratinocytes. This may account for the delay in terminal differentiation, and the weakened cohesion of the keratinocytes previously observed both in vivo and vitro.

Protease inhibitor therapy for recessive dystrophic epidermolysis bullosa: In vitro effect and clinical trial with camostat mesylate

Recently we reported that a kind of serine protease, SH protease, and collagenase might be involved in blister formation and, furthermore, that the cooperative action of these three proteases was essential for blister formation in recessive dystrophic epidermolysis bullosa. In this study we examined the inhibitory effect of clinically usable serine protease inhibitors for blister formation in organ culture and in clinical trials of recessive dystrophic epidermolysis bullosa patients. Camostat mesylate, a synthetic serine protease inhibitor that is available for the treatment of chronic pancreatitis, demonstrated a striking effect of inhibiting blistering in organ culture of normal human skin with recessive dystrophic epidermolysis bullosa blister fluids. Subsequently we administered camostat mesylate by topical application to four patients with recessive dystrophic epidermolysis bullosa to assess its ability to reduce blistering. Therapeutic response was favorable; a significant effect in decreasing the number of blisters was observed in three of four patients. These findings actually supported the hypothesis that a kind of serine protease had a close relationship with blistering in recessive dystrophic epidermolysis bullosa and that therapy with a clinically usable protease inhibitor was useful for the treatment of this disease.

Induction of an activation antigen on postcapillary venular endothelium in human skin organ culture.

We studied the effects of the immune mediators interleukin 1, interleukin 2, tumor necrosis factor, immune interferon, and lipopolysaccharide on the expression of the endothelial activation antigen recognized by the murine monoclonal antibody H4/18 in short term organ cultures of newborn foreskins. No endothelial staining was detectable before culture. Interleukin 1, tumor necrosis factor, and lipopolysaccharide each induced 2+ to 3+ H4/18 staining of microvascular endothelium at 6 hr. Combining mediators produced additive (3+ to 4+) effects, and reactivity was lost or markedly diminished by 24 hr. Incubation with culture medium alone resulted in 1+ to 2+ H4/18 staining at 6 hr, and medium conditioned by cultured foreskins, but not mock-conditioned medium, could induce H4/18 binding in cultured human umbilical vein endothelial cells. The spontaneous expression of microvascular staining in the foreskins was markedly inhibited by cyclosporin A, but not polymyxin B sulfate or dexamethasone; cyclosporin A did not inhibit induction of staining by exogenous mediators. Both light level and immunoultrastructural studies demonstrated H4/18 expression to be associated predominantly with postcapillary venular endothelial cells of the superficial vascular plexus. We conclude that microvascular endothelium of skin can undergo activation in response to exogenous and endogenous cytokines, with the greatest changes occurring in those portions of the vessels most involved in leukocyte and lymphocyte trafficking.

Dermo-epidermal blister formation by linear IgA dermatosis sera in normal human skin in organ culture.

AbstractFresh normal human skin samples were cultured at 37°C for 48 hours with sera from 3 patients with linear IgA dermatosis and 2 patients with dermatitis herpetiformis (DH). Dermo‐epidermal separation (DES) occurred in the skin samples cultured with the sera from linear IgA dermatosis patients, and linear IgA deposition was observed in the dermo‐epidermal junction zone. However, no IgA deposition or DES was observed in the skin cultured with the sera of DH patients or normal individuals.The results were the same when sera of patients with linear IgA dermatosis were heated at 56°C for 30 minutes before culturing with skin samples, but DES was prevented when a proteinase inhibitor, aprotinin (50%), or α2‐macroglobulin (10 mg/dl) was added to the cultures.Our results suggest that IgA deposition in the basement membrane and proteinases are involved in the formation of vesicles in patients with linear IgA dermatosis, but complements are not directly involved.

Cell surface and cytoskeletal changes associated with epidermal stratification and differentiation in organ cultures of embryonic human skin

Embryonic and fetal human epidermis differentiates in organ culture in an age dependent, though accelerated, manner. The older the specimen the less time is required for epidermal differentiation. Morphological markers of epidermal differentiation, including the different epidermal strata, keratohyalin granules, lamellar granules, and cornified cell envelopes, are formed in a manner that is faithful to development in vivo. The high molecular weight, “differentiation specific” (67 and 56.5 kDa) keratins are also expressed in these cultures, even in the absence of morphological evidence for keratinization. Unstratified, embryonic epidermis was found to stratify overnight in culture. The time course of cell surface changes, detected by lectin binding, and cytoskeletal changes, detected by expression of the high molecular weight keratins, was followed in these cultures. Peanut agglutinin (PNA) binding sites appeared overnight in culture coincidently with epidermal stratification while expression of the 67 and 56.5 kDa keratins was not detected until the third day of culture. The possible significance of these results is discussed.

Origin and properties of the blister formation factor in blister fluids from recessive dystrophic epidermolysis bullosa.

The origin and properties of the blister formation factor in recessive dystrophic epidermolysis bullosa (RDEB) blister fluids were investigated. Organ cultures of normal human skin incubated with RDEB dermis extract or with RDEB fibroblast culture medium (FCM) produced a clear subepidermal blister with histology similar to that of a RDEB blister in vivo. The injection of RDEB dermis extract into guinea-pig skin also induced dermal-epidermal separation with similar histology to the skin lesions of RDEB patients. The blister forming activity of RDEB FCM which induces the subepidermal blister was inactivated by heat (60 degrees C for 30 min), trypsin digestion and by treating with EDTA, EGTA, alpha 2-macroglobulin, diisopropyl fluorophosphate and N-ethylmaleimide, but was not affected by dialysis. These results suggest that the RDEB fibroblast produces a blister formation factor(s), and that blister formation may be caused by a combination of a metallo-protease, serine protease and SH protease.

Complement Fixation by Pemphigus Antibody. I. In Vitro Fixation to Organ and Tissue Culture Skin

Although complement is often detected in the intercellular substance of pemphigus skin lesions, the ability of pemphigus antibodies to fix complement in vitro is controversial. The purpose of this study was to test in vitro complement fixation abilities of pemphigus antibodies further using organ and tissue culture methods. Epidermal cell monolayers from mouse tail were incubated with the purified IgG fraction of pemphigus serum followed by purified Clq. Binding of Clq, as well as IgG was demonstrated by immunofluorescence methods. When purified Clq was replaced with normal human serum as a complement source, positive C3 and C4 staining were also evident. When purified IgG of normal human serum was used in place of pemphigus IgG, similar immunofluorescence staining was not observed. Further evidence for complement fixation in vitro by pemphigus antibodies was obtained using organ cultures. Organ culture of normal human skin and monkey esophageal mucosa cultured in purified pemphigus IgG showed intercellular substance binding of IgG. No binding was observed when normal IgG was substituted for pemphigus IgG. Additional organ culture sections were then treated with complement (fresh normal human serum) and tested by in vitro complement staining. Fixation of Clq, C4, and C3 was noted in intercellular substance areas of organ cultured skin and mucosa incubated with pemphigus IgG but not those incubated with normal IgG. Prior treatment of pemphigus IgG organ cultured skin sections with unlabeled anti-C3, blocked positive C3 staining. These results suggest that some pemphigus antibodies are capable of activating complement in vitro.

Epidermolysis bullosa simplex blister fluid induces an intra-epidermal blister in cultured normal skin.

Organ cultures of normal human skin incubated with blister fluid from a patient with epidermolysis bullosa simplex (EBS) produced a clean intra-epidermal blister with histology similar to that of an EBS blister.

Regulation of Gelatinase in Human Skin Organ Cultures by Glucocorticoids.

Regulation of Gelatinase in Human Skin Organ Cultures by Glucocorticoids.

Differential Distribution of Langerhans Cells in Organ Culture of Human Skin

Epidermal Langerhans cells (LCs) function as the antigen-presenting cells in such cutaneous cell-mediated immune responses as contact hypersensitivity and in the mixed epidermal cell-lymphocyte reaction. They have also been implicated in the immune response in skin allograft rejection. Since organ culture of thyroid and pancreas has been shown to prolong allograft survival, presumably through the loss of antigen-presenting cells, we examined the effect of skin explant culture on LC survival. Human skin explants were placed in organ culture and examined serially as whole mounts of epidermis for the presence of LCs as judged by ATPase activity, and OKT-6 and HLA-DR antigens. Although we observed morphologic changes and an absolute reduction in the number of positively stained cells, culture for up to 28 days failed to deplete explants of these cells. Langerhans cells were also sought in the epidermal outgrowths that develop peripheral to the original explants. They were never seen in the area beyond 0.3 mm from the explant edge. Organ culture of skin thus provides a means to explore the contribution of LCs to skin allograft rejection by comparing the immunogenicity of epidermal portions of the explant with the epidermal outgrowth.

Effects of sex steroids on human skin in organ culture

Histometric and autoradiographic methods were applied to investigate the actions of sex hormones on the cultured epidermis of post-menopausal women. Both testosterone and progesterone at the concentration of 10(-9)-10(-7) mol/l enhanced the keratinization of the epidermal cells during the 7-day culture. The numbers of the granular cells were increased by both of these hormones and the transit time of the epidermal cells was shortened by progesterone. Progesterone (but not testosterone) influenced cell proliferation, too, as indicated by an increased labelling of the epidermal cells with [3H]thymidine. In a contrast to other sex steroids, 17-beta-estradiol (10(-7) and 10(-9) mol/l) appeared to be ineffective in short-term organ culture.

Organ culture of adult human skin; effect of culture temperature.

ABSTRACTIn the organ culture of adult human skin, long‐term preservation of cultured epidermis was obtained when the culture temperature was shifted from 37°C to 32°C. The epidermal cell kinetics were compared for both culture temperatures using the findings of mitotic rate, mitotic distribution and autoradiographic labelling index with tritiated‐thymidine (3H‐TdR). As a result, it appeared that a lowered culture temperature controlled the cell cycle in vitro to maintain long‐term, active epidermal proliferation.

Effect of serum and oxygen tension on human skin organ culture: a histometric analysis

No abstract available