Extracts Of Normal Skin Research Articles

Harming Thyself

Psoriasis is an inflammatory autoimmune disorder of the skin characterized by excessive proliferation of keratinocytes and the presence of plasmacytoid dendritic cells (pDCs). Whereas pDCs normally sense microbial DNA in a Toll-like receptor 9 (TLR9)-dependent fashion, in psoriatic lesions pDCs respond to self-DNA and secrete interferon-α (IFN-α), which exacerbates the condition. Lande et al . investigated the mechanism involved and screened extracts of normal and psoriatic human skin (fractionated by reversed-phase high-performance liquid chromatography) for factors that could stimulate IFN-α production by cultured human pDCs. Extracts of normal skin did not induce a response; however, a fraction from psoriatic skin stimulated robust production of IFN-α as measured by enzyme-linked immunosorbent assay. Mass spectrometry identified the cathelicidin LL37, an antimicrobial peptide, as the stimulating factor. The addition of an antibody against LL37 blocked the stimulation by psoriatic skin extract of IFN-α secretion by pDCs. Western blotting and real-time reverse transcription polymerase chain reaction assays showed that LL37 protein and mRNA were more abundant in psoriatic skin than in healthy skin. LL37 is produced in the skin as an antibacterial defense after tissue damage, which often precedes psoriatic episodes. Treatment of pDC cultures with DNase, which removed DNA from dead pDCs, blocked the ability of LL37 to stimulate IFN-α secretion. Atomic force microscopy showed that LL37 formed aggregated complexes with DNA, and confocal microscopy and flow cytometry showed that these LL37-DNA complexes entered pDCs and were localized to early endosomes, where TLR9 is found. As Baumgarth and Bevins suggest, this study, which shows that LL37 can complex with self-DNA (released from dead keratinocytes) to activate pDCs in psoriasis and so disrupt tolerance (the nonresponsiveness to self) has implications for the treatment of autoimmunity. R. Lande, J. Gregorio, V. Facchinetti, B. Chatterjee, Y.-H. Wang, B. Homey, W. Cao, Y.-H. Wang, B. Su, F. O. Nestle, T. Zal, I. Mellman, J.-M. Schröder, Y.-J. Liu, M. Gilliet, Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature 449 , 564-569 (2007). [PubMed] N. Baumgarth, C. L. Bevins, Skin deep but complex. Nature , 449 , 551-553 (2007). [PubMed]

Expression of matrix metalloproteinase (MMP)‐2, MMP‐9, MMP‐13, and MT1‐MMP in skin tumors of human papillomavirus type 8 transgenic mice

Human papillomaviruses (HPV) are small DNA viruses that induce a wide variety of hyperproliferative lesions in cutaneous and mucosal epithelia. It is proposed that HPV is involved in non-melanoma skin cancer development. We have previously shown that HPV8 transgenic mice spontaneously develop papillomatous skin tumors. Histology revealed epidermal hyperplasia, acanthosis and hypergranulosis and in some cases squamous cell carcinomas (SCC). Zymographic and immunoblot analysis of normal skin extracts identified increased amounts of matrix metalloproteinase (MMP)-9, MMP-13 and MT1-MMP in HPV8-positive mice compared with HPV8-negative animals. In situ gelatin zymography of tumor specimens displayed a strong proteolytic activity in papillomas, and SCC putatively attributed to the increased amounts of activated MMP-9 found in tissue extracts. In addition, immunoblot analysis revealed increased amounts of active MMP-13 and MT1-MMP in tumor extracts as compared with control extracts. Immunohistochemical stainings of SCC specimens depicted MMP-13 to be specifically expressed in stromal fibroblasts neighboring the tumor islands, whereas MT1-MMP was detected both in tumor cells and in stromal cells. Taken together, these results implicate a role for MMPs in the development of HPV8-induced cutaneous tumors.

Differential expression of a human endogenous retrovirus E transmembrane envelope glycoprotein in normal, psoriatic and atopic dermatitis human skin

Psoriasis is a common inflammatory skin disease characterized by uncontrolled proliferation of keratinocytes and recruitment of T lymphocytes into the skin. The possible role of human endogenous retroviruses (HERVs) in the induction of psoriasis has been suggested, based upon the previous observations of retrovirus-like particles in psoriasis from skin lesional plaques, urine and stimulated lymphocytes. To investigate the expression of HERV-E transmembrane envelope glycoprotein (HERV-E env) in normal, psoriatic and atopic human skin, and to examine the influence of ultraviolet (UV) B irradiation on HERV-E env expression in normal human epidermal keratinocytes. The analysis was performed on both skin biopsies and organotypic skin cultures using immunofluorescence and Western immunoblotting. UVB irradiation (312 nm) of cultured normal human keratinocytes was performed using a dose of 30 mJ cm(-2). Positive staining was observed in most of the psoriatic and atopic skin samples, whereas only 15% of the normal skin samples were faintly positive. In addition, the pattern of expression of HERV-E env differed markedly in psoriasis vs. atopy. By Western blotting analysis, two main proteins of 54 and 57 kDa were detected in extracts of normal skin, normal keratinocyte cultures and reconstructed epidermis from psoriatic and normal punch biopsies. An increased level of expression of these proteins was noted in extracts from psoriatic vs. normal reconstructed epidermis. The overexpression of the 57-kDa protein in normal human cultured keratinocytes was dramatically reduced by UVB irradiation. These data suggest for the first time that HERV-E env is expressed in normal and pathological human skin. Further studies are now required to elucidate the role of such viral proteins in the pathogenesis of psoriasis.

Persistence of Both Peripheral and Non-Peripheral Corneodesmosomes in the Upper Stratum Corneum of Winter Xerosis Skin Versus only Peripheral in Normal Skin

To understand the biochemical abnormalities that underlie the reduced desquamation observed in dry skin, we analyzed corneodesmosome degradation in normal and winter xerosis skin. Western blotting of total proteins from corneocytes obtained by varnish-strippings from the legs of 56 volunteers with normal (26) or xerotic (30) skin was performed using antibodies specific for (corneo)desmosome proteins. In the whole population, the amounts of desmoglein 1 and plakoglobin were found to be correlated, but were not related to the amounts of corneodesmosin. This suggests simultaneous proteolysis for the former proteins differing from that of corneodesmosin. Neither entire desmoplakins nor any proteolysis-derived fragments were detected. The amounts of corneodesmosin, desmoglein 1, and plakoglobin detected were found to be significantly higher in xerotic compared with normal skin extracts. Conventional and freeze-fracture electron microscopy showed the absence of nonperipheral corneodesmosomes in the upper stratum corneum of normal skin but the presence of a significant number of these structures in the same layer of winter xerosis skin. These results provide a more precise description of the proteolysis of corneodesmosome components in the upper cornified layer of the epidermis. They support previous studies demonstrating the importance of corneodesmosome degradation in desquamation and reveal that the nonperipheral corneodesmosomes, which are totally degraded during maturation of the stratum corneum in normal skin, persist in winter xerosis, probably leading to abnormal desquamation.

Apolipoprotein E is Present in Primary Localized Cutaneous Amyloidosis

Apolipoprotein E (apoE) is one of the amyloid associated proteins that is found in the amyloid plaque of Alzheimer's disease and systemic amyloidosis. ApoE might play an important part in the etiology of Alzheimer's disease by functioning as a "pathologic chaperone" to promote the formation of amyloid filaments. In this study, we investigated whether apoE is associated with amyloid deposits of primary localized cutaneous amyloidosis using immunohistochemistry, immunogold electron microscopy, and immunoblotting. The subjects consisted of 12 patients with lichen amyloidosus and one patient with macular amyloidosis. Light microscopically, amyloid deposits in the dermal papillae were round in shape and stained with Congo red. Immunohistochemically, apoE was detected in amyloid deposits in all the cases examined. Immunogold electron microscopy showed apoE immunoreactivity on the amyloid deposition. Immunoblots of amyloid-positive skin showed 35K and 14K proteins, which were taken to be apoE and its fragment, respectively. In normal skin extract, only the 35K protein was detected by the anti-human apoE. Moreover, the intensity of the amyloid-positive skin sample was stronger than that of the normal skin sample. Monoclonal anti-cytokeratin antibody reacted with the 45K protein of the amyloid-positive skin extract. These results indicate that apoE is a component of primary localized cutaneous amyloidosis, and that it might play an important role in primary localized cutaneous amyloidosis.

Surprisingly high levels of anaphylatoxin C5a des Arg are extractable from psoriatic scales

Water-soluble extracts from psoriatic scales and normal human skin were prepared using either phosphate-buffered saline, pH 7.2, or 0.1 M carbonate buffer, pH 10.8. Anaphylatoxin C5a des Arg was quantified using a novel sandwich enzyme-linked immunosorbent assay (ELISA) using neoepitope-specific monoclonal antibodies. Alkali was about five to eight times more efficient than PBS in extracting C5a des Arg from scales, probably via dissociation of bound C5a des Arg. C5a des Arg concentration in scales from three patients suffering from psoriasis vulgaris varied between 2.5 and 4.6 ng/mg scale. No C5a des Arg was detectable in normal skin extracts. The biological activity of alkali-extractable C5a des Arg, i.e. chemotaxis, was preserved. The concentration of C5a des Arg relative to the concentration of albumin was taken as a parameter of the degree of complement activation in the psoriatic lesions, and was found to be more than six times higher than values attained in serum after maximum complement activation by zymosan. We conclude that complement activation may play a quantitatively important role in the inflammatory process in psoriasis.

Identification of Basement Membrane Zone Antigens Defined by Antibodies that React to Both the Epidermal and Dermal Side of 1 M Sodium Chloride Split Skin

Some individuals have basement membrane zone (BMZ) antibodies that react to both the epidermal and dermal side of skin split with 1 M NaCl. To examine the significance of this combined staining pattern, we tested sera from 185 different, sequential, patients with BMZ antibodies for reactivity to normal human skin split with 1 M NaCl. Six sera (3.2%) stained both the epidermal and dermal sides of split skin, 173 (93.5%) stained only the epidermal side, and 6 (3.2%) only the dermal side. All six sera with a combined staining pattern yielded the same pattern when tested with three different specimens of skin, indicating that this pattern is reproducible. By immunoblot analysis, five (83%) of the six combined staining sera reacted to a 160-kD antigen present only in epidermal extracts of normal skin, one reacted in addition to a 230-kD epidermal antigen, and one did not react to either epidermal or dermal extracts. In contrast, five (83%) of the six sera with dermal staining reacted to a 290-kD antigen present only in dermal extracts. Eighteen (90%) of twenty representative epidermal staining sera reacted to a 230-kD epidermal antigen and seven (35%) sera (five with the 230-kD antibody and two without) also reacted to the 160-kD epidermal antigen. Affinity purified antibody to the 160-kD antigen defined by combined staining sera reacted to the BMZ of normal human skin. These results indicate that the combined staining pattern on 1 M NaCl split skin is due to the presence of a distinctive antibody response directed predominantly to a 160-kD BMZ antigen located on the epidermal side of the split skin and to an as yet unidentified BMZ antigen located on the dermal side.

Western blot analysis of the antigen in pemphigoid gestationis

Using an immunoblotting technique, sera from 25 patients with pemphigoid gestationis were examined and tested against epidermal and dermal extracts of normal skin. The major antigen recognized by seven patients' sera was a molecule of 180 kDa, pemphigoid gestationis antigen, extractable only from the epidermis. Sera from 18 patients with bullous pemphigoid were studied as positive controls and the major antigen recognized was a larger molecule of 220 kDa. There was some degree of shared recognition of antigens with three patients with pemphigoid gestationis recognizing the 220 kDa bullous pemphigoid antigen. In addition one bullous pemphigoid serum recognized the 180 kDa pemphigoid gestationis antigen. The dominant pemphigoid gestationis antigen, however, differs from bullous pemphigoid antigen.

Chemotactic factors for macrophages produced in inflamed skin.

A newly discovered macrophage-chemotactic factor (MCFS-1) was extracted and purified (488-fold) from skin sites of guinea pigs showing delayed hypersensitivity to bovine gamma-globulin. MCFS-1 is a heat-labile glycoprotein with a molecular weight of 150,000, consisting of two polypeptide chains. It has in vivo as well as in vitro chemotactic activity for macrophages and shared 50% of the chemotactic activities for macrophages in the extract. It is produced by limited proteolysis of endogenous trypsin-like protease(s) of plasma precursor protein (Pre-MCFS-1). Enzyme immunohistochemistry reveals that Pre-MCFS-1 is produced in the liver and distributed in the normal skin as a proinflammatory factor. ELISA reveals that the concentrations of the factor are: 140 micrograms/ml in plasma, 7.1 micrograms/ml in inflamed skin extract, and 2.8 micrograms/ml in normal skin extract.

Identification of Collagen Fibrils in Scleroderma Skin

Skin from early and late stages of scleroderma has been shown to contain large amounts of thin (30-40 nm diameter) collagen fibrils that may be present in bundles or intermingled with large diameter fibrils (90-120 nm). The nature of these fibrils is unknown. Skin biopsies were obtained from involved areas of nine patients with progressive systemic sclerosis (PSS), one case of generalized morphea, one case of morphea, and six normal controls. Intact skin was analyzed by immunoelectron microscopy (IEM), while extracts were subjected to sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE), Western immunoblotting, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA). Fine fibrils 20-40 nm in diameter in the mid to lower dermis of scleroderma skin were labeled with antibodies directed against the aminopropeptide (AP) of type III procollagen. Antibodies directed against the AP of type I procollagen labelled fine fibrils in the lower dermis. Larger fibrils (80-120 nm) did not label. pN alpha 1 (III) was found to be present in both normal and scleroderma skin. Extracts of scleroderma skin contained 2.5 times the amount of pN (III) collagen and 3.0 times the amount of fibronectin as did extracts of normal skin. The data indicate that the increase in thin fibrils in scleroderma skin is most likely due to an increase in type III collagen, which retains the AP at its surface.

Mechanism of acute depletion of plasma fibronectin following thermal injury in rats. Appearance of a gelatinlike ligand in plasma.

Plasma fibronectin was depleted within 15 min following sublethal burn, followed by partial recovery at 8 h and complete restoration by 24 h in anesthetized rats. Radiolabeled 75Se-plasma fibronectin, injected intravenously before burn, was rapidly sequestered in burn skin as well as the liver. Fibronectin levels at 2 h postburn as detected by immunoassay vs. 75Se-plasma fibronectin indicated that more fibronectin was in the plasma than detected by electroimmunoassay. Crossed immunoelectrophoretic analysis of fibronectin in early postburn plasma demonstrated a reduced electrophoretic mobility of the fibronectin antigen. Addition of heparin or fibrin, both of which have affinity for fibronectin, to normal plasma was unable to reproduce this altered fibronectin electrophoretic pattern. In contrast, addition of gelatin or native collagen to normal plasma reproduced the abnormal electrophoretic pattern of fibronectin seen in burn plasma. Extracts of burned skin, but not extracts of normal skin, when added to normal plasma, elicited a similar altered electrophoretic pattern for fibronectin. By gel filtration, fibronectin in burn plasma had an apparent molecular weight approximately 40% greater than that observed in normal plasma. These data suggest the release into the blood of a gelatinlike ligand from burned skin, which complexes with plasma fibronectin. Thus, fibronectin deficiency acutely postburn appears mediated by (a) its accumulation at the site of burn injury; (b) its removal from the circulation by the liver; and (c) its presence in the plasma in a form that is less detectable by immunoassay.

‘Metabolic’ factors in thermal injury: role of nucleic acids

We believe that toxic events observed after thermal injury may be caused by the release of normally intracellular substances into the circulation. We define these substances as ‘metabolic’ factors. Analysis of extracts prepared from normal and burned mouse skin indicates that the burned skin extract contains increased clot-promoting (Thromboplastin-like) substances and, perhaps, less RNA than normal skin extracts. Injection of RNA or its breakdown products into the burned site significantly increases the acute mortality in burned mice. No increase in mortality is observed when these substances are injected into a non-burned site on burned mice. We suggest that (Thromboplastin-like substances and RNA or RNA breakdown products may be some of the ‘metabolic’ factors involved in acute burn toxicity. Upon being released from their intracellular residence after thermal injury, their combined activity contributes to the acute mortality observed.

Immunity to melanoma associated antigens detected by both leukocyte adherence inhibition and antibody dependent cellular cytotoxicity assays

Two different assays, leukocyte adherence inhibition (LAI) and antibody dependent cellular cytotoxicity (ADCC) have been used to measure the immune responses of 69 melanoma patients, 116 patients with other tumours and 64 normal controls to a number of melanoma and control antigens. Using the LAI test, melanoma patients were significantly more reactive (43-69% positive) than normal controls (7-32% positive) to membrane extracts from 3 to 6 melanoma cell lines, and 4 to 6 extracts of melanoma biopsy specimens. However, the proportion of patients with other tumours reacting with 3 of these extracts was similar to melanoma patients. Melanoma patients were more reactive to the melanoma extract than to extracts of normal skin, normal muscle and 2 breast cancer cell lines. ADCC tests were used to detect anti-melanoma antibodies in patient sera. Preferential reactivity by melanoma patients was detected towards only 2 of the 6 melanoma cell lines tested--53% of 55 melanoma sera reactive to LiBr compared with 28% of 79 sera from patients with other tumours and 17% of 29 normal sera (P less than 0.05); 42% of 31 melanoma sera reactive to MM127 compared with 18% of 22 sera from other tumour patients (n.s.) and 5% of 20 normal sera (P less than 0.005). Melanoma patients tested against a number of melanoma cell lines by ADCC or antigen extracts by LAI generally reacted with one or more, but not all of them. Thus, incomplete cross-reactivity between different melanomas was observed. There was no correlation between results of the same patient in the 2 tests.

Growth of Pseudomonas aeruginosa in normal and burned skin extract: role of extracellular proteases.

Growth curves and mean generation times (MGT) were determined for Pseudomonas aeruginosa strain M-2 (protease +) and strain PA-103 (protease +/-) in burned skin extract (BSE) and in normal skin extract (NSE). Strain M-2 grew on NSE or BSE with an MGT of 30 min. Strain PA-103 grew in NSE at a similar MGT; however, PA-103 in BSE had a MGT of 65 min. When protease was added to BSE, PA-103 grew as rapidly as M-2. When ammonium sulfate was added to inhibit protease production, the MGT of M-2 slowed to that of M2 in both BSE in NSE. The MGT of PA-103 in amino acid-supplemented BSE was similar to that of PA-103 in BSE. The MGT of PA-103 in amino acid-supplemented BSE was similar to that of M-2 in both BSE andNSE. These data suggest that protease may serve as a virulence factor by modifying the available nutrients in burned skin. As a result, nutrients are formed that permit an enhanced growth rate and amore rapid establishment of the infection in the host.

QUANTITATION AND IMMUNOCYTOCHEMICAL LOCALIZATION OF HUMAN SKIN COLLAGENASE IN BASAL CELL CARCINOMA

Human skin collagenase was quantitated by radioimmunoassay in 21 basal cell carcinomas. Immunoreactive collagenase protein was found to be approximately 2-fold greater in extracts of these tumors than in extracts of normal skin, suggesting that this enzyme may be important in the pathogenesis of soft tissue destruction in vivo. To further define the role of collagenase in such destruction, immunofluorescent staining with specific antiserum to human skin collagenase was used to localize collagenase in the basal cell carcinomas. The enzyme was found only in the stromal elements surrounding the tumor islands. No staining of the epithelial components of the basal cell carcinomas was found. These findings suggest that the normal connective tissue elements may have been stimulated to produce an increased amount of collagenase and emphasize the importance of epithelial-stromal interaction in soft tissue invasiveness.

Wart-Associated Antigens. I. Isolation of Tissue Antigens Using Antibody Immunoadsorbents

Two antigens of human wart tissue, a nuclear and a cell surface antigen, were isolated from whole tissue homogenates by elution from antibody-Sepharose immunoadsorbents. Neither antigen was a structural component of purified human papilloma virus. The antigens were detected by immunofluorescence in warts, squamous cell carcinomas, fetal skin, and psoriatic epidermis, but not in normal skin or basal cell carcinomas. They could be detected in concentrated extracts of normal skin by agar gel diffusion. The antigens belong to the growing class of substances that are readily detected in tumors and in fetal tissues, but are present only in minute quantities in normal adult tissue.

Collagen made of extended -chains, procollagen, in genetically-defective dermatosparaxic calves.

Collagen has been extracted from the skin of dermatosparaxic calves and fractionated by acrylamide-gel electrophoresis and CM-cellulose chromatography. This collagen contains in large proportion two additional types of α-chains, P-α1 and P-α2. The first, P-α1, ressembles α1, of a collagen extracted from the skin of a normal calf in its amino-acid composition but its molecular weight is 10% larger. The second, P-α2, is similar to a normal α2 in its amino-acid composition but its molecular weight is increased by 5%. The abnormal α-chains contain six to eight half cystine residues in p-α1 and one in p-α2. As seen under the electron microscope on segment long spacing, the dermatosparaxic collagen is 25 nm larger than the normal collagen and the in creased length results from extension of the polypeptide chain at the N-terminal extremity of the molecules. Although the collagen is less extractable from the dermatosparaxic skin than from the normal skin and dimers and other polymers of the abnormal α-chains are observed, the dermatosparaxic collagen does not, in vitro, form fibrils of normal stability. Since an enzyme activity which can excise the N-terminal extension peptide of the dermatosparaxic collagen has been identified in extracts of normal skin but is absent in the dermatosparaxic animals, it is suggested that the dermatosparaxic collagen is a form of “procollagen”, from which all or a part of these peptides have not been removed and that such peptides function as coordinators in the post synthesis assembly of the collagen α-chains.

Epidermal chalone and mitotic control in the Vx2 epidermal tumour.

In a number of tissues It has been found that mitotic activity is controlled by specific mitotic inhibitors, called chalones. It is now shown that in four malignant tumours (epidermal carcinoma, granu-locytic leukaemia, melanotic and amelanotic melanomata) the chalones of the tissues of origin are still synthesized, although the chalone content is abnormally low, and that in all cases the tumours are still capable of responding by mitotic inhibition when the chalone content is artificially raised. It is also shown that when extracts of normal skin or of melanomata (both of which contain the melanocyte chalone) are injected for 5 days into mice or hamsters bearing large malignant melanomata, rapid tumour regression occurs and in some cases leads to a complete cure.

Immunologic Studies in Thermal Injury: Antigenic Properties of Burned Guinea Pig Skin

By means of rabbit immune sera, an antigenic component characteristic for burned guinea pig skin was demonstrated by double diffusion gel precipitation tests. This antigen could not be detected in normal skin extract or in skin burned after its removal. Evidence was presented that the antigen characteristc for the burned skin is identical with or closely related to a normal component of guinea pig plasma, presumably fibrinogen.

Soluble proteins of a melanoma and normal skin from the swordtail, platyfish, and their hybrids.

Acrylamide gel electrophoresis has been used to make comparisons of the distribution of soluble protein and the activity of esterase in skin and melanoma extracts of the swordtail, the platyfish, and their hybrids. There is a striking difference between the extracts of normal skin and tumor tissue derived from the same cytological elements. Several prominent protein bands are found only in the tumor tissue. Their origin and function are unknown.