Effects of platelet derived growth factor (PDGF) on fibronectin (FN) production by human skin and scar fibroblasts

The fibroblast-type cell found in hypertrophic scars and keloids demonstrates an elevated fibronectin (FN) production, compared to the same type of cell in normal dermis. We wished to determine if the effects of platelet derived growth factor (PDGF) on FN production in these cell types would be equivalent or different. Cell lines were established from the dermis (reticularis) of hypertrophic scars, keloids, uninvolved normal skin adjacent to the lesions, including an assumed normal skin adjacent to a keloid (AS), and normal skin from a different uninjured patient (DS). Each parent tissue from which the cell lines originated was diagnosed histologically. Each hypertrophic scar, keloid and normal adjacent skin, with one exception, showed typical histologic findings confirming the clinical diagnosis. DS was also normal. AS, although assumed to be normal, in fact, demonstrated portions of nodules from the adjacent keloid. All cell lines were grown under standard conditions with subconfluent cells metabolically labeled for radioimmunoassays measuring FN at passage 3 (8 to 9 weeks in culture) in the absence and presence of PDGF. Significant differences in production of FN/cell and FN/PR/cell between two hypertrophic scars and their matched normal skins and for one keloid and its matched normal skin were observed. However, no significant difference was observed between the other keloid and AS, nor between the other hypertrophic scar and DS. PDGF significantly stimulated FN production in 2 of 4 NS cell lines, and in the AS cell line. By FN/cell values, 2 of 5 cell lines from the lesions were inhibited and one was increased. In terms of FN/PR/cell, 1 of 5 cell lines from the lesions was stimulated and the others showed no differences. The mixed results may be attributable to the likelihood that the cell lines represent mixed populations. This study demonstrates the importance of: 1) histological characterization of all parent tissues from which cell lines are derived, and 2) matching cell lines from lesions with cell lines from uninvolved normal dermis, in the same individual.     

Measurement of collagen-proteoglycan interaction in hypertrophic scars

Collagen-proteoglycan interaction in hypertrophic scars was measured by staining tissue collagen with Sirius red, followed by elution of the bound stain, and photometric quantitation before and after papain digestion. Control sections from each of the specimens were treated with buffer solution instead of papain. The difference in dye concentration observed between respective untreated and digested sections was considered to be due to the unmasked basic groups of collagen originally bound to proteoglycans. The results suggest a significantly increased collagen-proteoglycan interaction in human hypertrophic scars as compared with nonhypertrophic scars, normal skin, and granulation tissues.     

慢性湿疹患者皮损、鼻粘膜需氧微生物群的定位、定量研究

本文首次应用定位、定量方法对23例小腿部慢性湿疹患者皮损、邻近皮肤及鼻孔的菌群,从微生态学的角度进行探讨,并将结果同24例正常人小腿皮肤及鼻孔的菌群做了比较。其结果表明:金黄色葡萄球菌为皮损及邻近皮肤的主要菌种,并且其分离率及密度有按正常皮肤、邻近皮肤、皮损依次增高现象;类白喉杆菌的数量在皮损部位明显低于正常皮肤与邻近皮肤;表皮葡萄球菌分离率皮损及邻近皮肤均明显降低。本组结果同正常对照组相比其皮肤微生物群菌谱虽无不同,但其数量的变异是显著的,这对探讨湿疹的病因及用生态制剂等调整皮肤菌群使其恢复微生态平衡达到防治目的有所裨益。     

Characterization of the collagen of human hypertrophic and normal scars.

The collagen produced in response to an injury of human skin is initially stabilized by a cross-link derived from hydroxyallysine, and characteristic of embryonic skin. In normal healing there is a change over with time to the cross-link derived from allysine, which is typical of young skin collagen. In contrast, hypertrophic scars fail to follow the time-related changes of normal skin, but retain the characteristics of embryonic collagen, indicating a continued rapid turnover of the collagen. This is further supported by the high proportion of the embryonic Type III collagen present in hypertrophic scars.     

瘢痕疙瘩中TIEG1的高表达

转化生长因子-β1(TGF-β1/Smads)信号转导通路的持续激活是瘢痕疙瘩形成的重要机制.研究发现这条通路重要的负反馈调节信号分子Smad7表达明显下调,Smad2/3的磷酸化水平和蛋白质量并无明显改变.但是,Smad7下调的机制尚不清楚.采用生物信息学方法对Smad7的启动子进行分析;用RT-PCR和蛋白质印迹分别检测了正常皮肤、正常瘢痕及瘢痕疙瘩组织中的Sp1样转录因子TIEG1mRNA及蛋白质的表达水平;体外培养正常皮肤、正常瘢痕及瘢痕疙瘩成纤维细胞,检测TIEG1 mRNA及蛋白的表达水平.研究结果显示,Smad7启动子上有Sp1的位点,TIEG1 mRNA及蛋白质水平在瘢痕疙瘩组织及瘢痕疙瘩成纤维细胞中表达明显高于正常瘢痕和正常皮肤(P<0.05).说明瘢痕疙瘩中TIEG1可能是Smad7下调的重要原因,有必要进一步研究TIEG1对Smad7的调控作用机制.     

Tissue alpha-globulins in keloid formation.

The deposition of alpha-1-antitrypsin and alpha-2-macroglobulin, both known to be inhibitors of human skin collagenase, is significantly increased in keloids and in hypertrophic scars (as compared to normal skin). However, following intralesional triamcinolone treatment, a marked resorption of these abnormal scars occurs along with a significant reduction of the alpha-1-antitrypsin deposits. These findings suggest that alpha-globulins are involved in abnormal scar formation, and that triamcinolone may remove collagenase and/or protease inhibitors--thereby allowing activation of the collagenase with subsequent breakdown and resorption of the excessive collagen.     

Changes in the secretion and glycosylation of fibronectin by human skin fibroblasts associated with tuberous sclerosis

Fibroblasts from skin and skin lesions of patients with tuberous sclerosis (TS) and from skin of normal individuals were grown in culture. ELISA showed that the spent medium of those derived from TS skin lesions contained significantly more fibronectin (FN) than spent medium from the other cells. Amino acid compositional analysis of the FN from TS and normal sources revealed no substantial differences. However the FN of fibroblasts from TS-skin lesions was shown by HPAEC to contain a two- to three-fold increased content of carbohydrate. The changed monosaccharide composition was consistent with an increased content of N- and O-linked glycans and with the former containing polylactosamine chains. Fibroblasts from a normal individual were shown to proliferate more slowly and to produce larger cells when grown on FN from a TS skin lesion compared to growth on FN from normal skin. This revised version was published online in November 2006 with corrections to the Cover Date.     

Is disturbed clearance of apoptotic keratinocytes responsible for UVB-induced inflammatory skin lesions in systemic lupus erythematosus?

Apoptotic cells are thought to play an essential role in the pathogenesis of systemic lupus erythematosus (SLE). We hypothesise that delayed or altered clearance of apoptotic cells after UV irradiation will lead to inflammation in the skin of SLE patients. Fifteen SLE patients and 13 controls were irradiated with two minimal erythemal doses (MEDs) of ultraviolet B light (UVB). Subsequently, skin biopsies were analysed (immuno)histologically, over 10 days, for numbers of apoptotic cells, T cells, macrophages, and deposition of immunoglobulin and complement. Additionally, to compare results with cutaneous lesions of SLE patients, 20 biopsies of lupus erythematosus (LE) skin lesions were analysed morphologically for apoptotic cells and infiltrate. Clearance rate of apoptotic cells after irradiation did not differ between patients and controls. Influx of macrophages in dermal and epidermal layers was significantly increased in patients compared with controls. Five out of 15 patients developed a dermal infiltrate that was associated with increased epidermal influx of T cells and macrophages but not with numbers of apoptotic cells or epidermal deposition of immunoglobulins. Macrophages were ingesting multiple apoptotic bodies. Inflammatory lesions in these patients were localised near accumulations of apoptotic keratinocytes similar as was seen in the majority of LE skin lesions. In vivo clearance rate of apoptotic cells is comparable between SLE patients and controls. However, the presence of inflammatory lesions in the vicinity of apoptotic cells, as observed both in UVB-induced and in LE skin lesions in SLE patients, suggests that these lesions result from an inflammatory clearance of apoptotic cells.     

Is disturbed clearance of apoptotic keratinocytes responsible for UVB-induced inflammatory skin lesions in systemic lupus erythematosus?

Apoptotic cells are thought to play an essential role in the pathogenesis of systemic lupus erythematosus (SLE). We hypothesise that delayed or altered clearance of apoptotic cells after UV irradiation will lead to inflammation in the skin of SLE patients. Fifteen SLE patients and 13 controls were irradiated with two minimal erythemal doses (MEDs) of ultraviolet B light (UVB). Subsequently, skin biopsies were analysed (immuno)histologically, over 10 days, for numbers of apoptotic cells, T cells, macrophages, and deposition of immunoglobulin and complement. Additionally, to compare results with cutaneous lesions of SLE patients, 20 biopsies of lupus erythematosus (LE) skin lesions were analysed morphologically for apoptotic cells and infiltrate. Clearance rate of apoptotic cells after irradiation did not differ between patients and controls. Influx of macrophages in dermal and epidermal layers was significantly increased in patients compared with controls. Five out of 15 patients developed a dermal infiltrate that was associated with increased epidermal influx of T cells and macrophages but not with numbers of apoptotic cells or epidermal deposition of immunoglobulins. Macrophages were ingesting multiple apoptotic bodies. Inflammatory lesions in these patients were localised near accumulations of apoptotic keratinocytes similar as was seen in the majority of LE skin lesions. In vivo clearance rate of apoptotic cells is comparable between SLE patients and controls. However, the presence of inflammatory lesions in the vicinity of apoptotic cells, as observed both in UVB-induced and in LE skin lesions in SLE patients, suggests that these lesions result from an inflammatory clearance of apoptotic cells.     

Comparative study of Langerhans cells in normal and pathological human scars. II. Hypertrophic scars.

Langerhans cells (LCs) seem to play a crucial role in the immune system of the skin. Changes in their density, distribution, phenotype and/or morphology have been described in a number of skin diseases, mostly immunologically mediated. For this reason, we investigated LCs in human hypertrophic scars, since these scars are presently believed to have an immunological basis. A preliminary analysis of the histological features was carried out on vertical serial sections, stained with hematoxylin and eosin. Both epidermal and dermal components of hypertrophic scar biopsies were examined. The total epidermal thickness and the thickness of the single epidermal layers were also measured; the values obtained were similar to those of control skin and normotrophic scars. Subsequently, CDla-positive LCs, revealed by indirect immunofluorescence and immunoperoxidase techniques, were studied to determine their position among the epidermal layers and within the dermis, their dimensions, their density and their morphology. According to these observations, two main types of hypertrophic scars were identified. In the first type (7 scars), LCs were widely clustered within both the whole epidermis and the dermis. Their density was increased (about 750 cells/mm2 of epidermal area), if compared to control skin and normotrophic scars (both about 400 cells/mm2 of epidermal area; p less than 0.001). The epidermal cell profiles, nearly three times larger than those of control skin, exhibited a dense network of interconnected dendrites. Further analysis for the presence of HLA-DR molecules revealed an anomalous expression of these antigens on keratinocytes. In the second type (3 scars), LCs density within the stratum Malpighii was unchanged, relative to control skin and normal scars, while CDla-positive cell bodies remained numerous in basal position and within the subpapillary corion. Epidermal LCs, only slightly larger than those evidentiated in control skin, displayed short and retracted dendritic projections. The aberrant expression of HLA-DR antigens on keratinocytes was very weak and sparse. The present results strongly suggest an immunologically activated state of the tissues examined; they provide morphological data that support the involvement of the immune system in hypertrophic scarring.     

病理性瘢痕中主要氧化酶和抗氧化酶活性测定

采用化学比色法测定正常皮肤(8例)、增生性瘢痕(10例)及瘢痕疙瘩(10例)组织中黄嘌呤氧化酶(xanthine oxidase,XO)、铜锌超氧化物歧化酶(copper,zinc-superoxide dismutase,CuZn-SOD)、过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase,GPX)活性以及丙二醛(malonaldehyde,MDA)含量.结果表明:与正常皮肤比较,病理性瘢痕中XO和CuZn-SOD活性增加、CAT活性降低(P<0.05)而GPX活性不变,CAT/CuZn-SOD和GPX/CuZn-SOD活性比率下降(P<0.05),同时MDA含量升高(P<0.05).增生性瘢痕、瘢痕疙瘩之间比较均无差异.上述结果表明,在病理性瘢痕中,氧化酶XO,抗氧化酶CuZn-SOD、CAT以及GPX的活性改变可能是引起活性氧水平升高的原因之一,在抗氧化剂选择上,CAT可能较为合理.     

Comparative study of Langerhans cells in normal and pathological human scars. I. Atrophic scars.

In the present study, we investigated Langerhans cells (LCs) in the epidermal component of human atrophic scars, comparing them with those in control skin and normotrophic scars. A preliminary analysis of the histological features was first carried out on vertical serial sections, stained with hematoxylin and eosin. The total epidermal thickness and the thickness of the single epidermal layers were then measured, by means of a digitizing tablet and a morphometric program run on an Apple IIe computer. These parameters were found to be significantly lower (40%) in atrophic scars, if compared to control skin and normotrophic scars (p less than 0.05). CDla-positive and HLA-DR-positive LCs were marked by indirect immunofluorescence. Their position among the epidermal layers, their dimensions, their density and their morphology were examined. In atrophic scars, LCs were densely and evenly distributed in all the epidermal layers. Their density was increased (about 1200 cells/mm2 of epidermal area), if compared to control skin and normotrophic scars (both 300-400 cells/mm2 of epidermal area; p less than 0.001). The CDla-positive definite cell bodies, exhibiting an unstained nucleus, were as large as those evidentiated in the normotrophic scars and twice as much the control skin values (p less than 0.001). The present results provide morphological data that distinguish atrophic scars from control skin and normotrophic scars, and suggest an involvement of the Langerhans cells in this particular case of pathological scarring.     

Reorganization of skin scars on the back of rats]

The method of dosed mechanical injuries (DMI) when applied repeatedly to the mature skin scars in the rat's backs leads to their reduction and transformation into the regenerates having the structure resembling that of the normal skin. The more is the number of applications of DMI--the more is the amount of transformed scars.     

Matrix metalloproteinase‐2 and ‐9 activities in human keloids,hypertrophic and atrophic scars: a pilot study

Proteolytic degradation of extracellular matrix is one of the principal features of cutaneous wound healing but little is known about the activities of gelatinases; matrix metalloproteinase‐2 (MMP‐2) and matrix metalloproteinase‐9 (MMP‐9) on abnormal scar formation. The aim of this study is to determine collagen levels and the gelatinase activities in tissue from hypertrophic scars, atrophic scars, keloids and donor skin in 36 patients and 14 donors. Gelatinase levels (proenzyme + active enzyme) were determined by ELISA and their activities by gelatin zymography. MMP‐9 activity was undetectable in gelatin zymography analysis. Pro‐MMP‐2 levels (median) were highest in normal skin group 53.58 (36.40–75.11) OD µg^?1 protein, while active MMP‐2 levels were highest in keloid group 52.53 (42.47–61.51) OD µg^?1 protein. The active/pro ratio was the highest in keloid group 0.97 followed by hypertrophic scar, normal skin and atrophic scar groups 0.69 > 0.54 > 0.48, respectively. According to results of our study, the two‐phase theory of the duration of hypertrophic scar and keloid formation can be supported by the data of tissue collagen and gelatinase analysis. This study is the first to relate scar formation relationship in regard to gelatinase activation ratio in a keloid, hypertrophic and atrophic scar patient group which is chosen appropriate in age and sex. Copyright © 2009 John Wiley & Sons, Ltd.     

Circulating immune complexes in human breast cyst fluids: relationship with intracystic immunoglobulin and electrolyte levels

Circulating immune complexes, the major classes of immunoglobulins and electrolyte concentrations were measured in sixty-two breast cyst fluids aspirated in women affected by gross cystic breast disease. Two main classes of cysts were defined according to the Na/K ratio. Appreciable levels of immunoglobulins were found in almost all samples examined; 66% of breast cyst fluids showed increased levels of immune complexes. A highly significant linear correlation between increased values of immune complexes and immunoglobulin M (p less than 0.001) was found in apocrine cysts, characterized by Na/K ratio less than 3. However, a significant inverse linear correlation was found between positive values of immune complexes and lowered levels of immunoglobulins A (p less than 0.001) and G (p less than 0.001) in epithelial cysts with Na/K ratio greater than 3. These data suggest and confirm that the menstrual cycle can also influence or modulate the metabolic activity of human breast cells as a part of the secretory immune system. The relationship between immune complexes and immunoglobulins and electrolyte profiles may provide further knowledge about the immunological features of breast cyst fluid and suggest the possible alteration of immune-response in cystic breast lesions associated with increased cancer risk.     

Septic Gonococcal Dermatitis

The overall incidence in gonorrhoea of septic gonococcal dermatitis was found to be 1·9% (3% for the females and 0·7% for the males). In 23 patients the common presenting symptoms were arthritis or arthralgia and bouts of fever, but the characteristic skin lesions served as an early clue to the diagnosis, and Neisseria gonorrhoeae was isolated from the genitourinary tract or from the blood. With the use of immunofluorescent techniques gonococci were also found in smears prepared from the skin lesions. An immune response to gonococci was found with the complement fixation technique in 90% of the patients. The response to treatment with penicillin was prompt, with complete relief from joint pains and fever, usually within two to seven days. The skin lesions faded within a few days, but scars could be observed for up to four weeks.     

Abrogation of transforming growth factor-beta signaling by SMAD7 inhibits collagen gel contraction of human dermal fibroblasts

Human fibroproliferative disorders like hypertrophic scarring of the skin are characterized by increased contractility and excess extracellular matrix synthesis. A beneficial role of transforming growth factor (TGF)-beta in wound healing was proposed; however, chronic stimulation by this cytokine leads to fibrosis. In the present report, the intracellular TGF-beta signaling in fibroblasts derived from hypertrophic scars and normal skin was examined. In an attempt to intervene in profibrogenic TGF-beta functions, ectopic expression of Smad7 or dominant negative Smads3/4 completely inhibited contractility of scar-derived and normal fibroblasts after suspension in collagen gels. Both cell types displayed constitutive Smad2/3 phosphorylation and (CAGA)9-MLP-Luc activity with expression and phosphorylation of Smad3 being predominant in hypertrophic scar-derived fibroblasts. Down-regulation of intrinsic signaling with various TGF-beta antagonists, e.g. soluble TGF-beta receptor, latency-associated peptide, and anti-TGF-beta1 antibodies, confirms autocrine TGF-beta stimulation of both cell populations. Further, Smad7 expression inhibited alpha1 (I) collagen and alpha-smooth muscle actin expression. In summary, our data indicate that autocrine TGF-beta/Smad signaling is involved in contractility and matrix gene expression of fibroblasts from normal and hypertrophic scars. Smad7 inhibits these processes and may exert beneficial effects on excessive scar formation.     

Investigation of the influence of keloid-derived keratinocytes on fibroblast growth and proliferation in vitro

Keloids are disfiguring, proliferative scars that represent a pathological response to cutaneous injury. The overabundant extracellular matrix formation, largely from collagen deposition, is characteristic of these lesions and has led to investigations into the role of the fibroblast in its pathogenesis. Curiously, the role of the epidermis in extracellular matrix collagen deposition of normal skin has been established, but a similar hypothesis in keloids has not been investigated. The aim of this study was to investigate the influence of keloid epithelial keratinocytes on the growth and proliferation of normal fibroblasts in an in vitro serum-free co-culture system. A permeable membrane separated two chambers; the upper chamber contained a fully differentiated stratified epithelium derived from the skin of excised earlobe keloid specimens, whereas the lower chamber contained a monolayer of normal or keloid fibroblasts. Both cell types were nourished by serum-free medium from the lower chamber.Epithelial keratinocytes from five separate earlobe keloid specimens were investigated. Four sets of quadruplicates were performed for each specimen co-cultured with normal fibroblasts or keloid-derived fibroblasts. Controls consisted of (1) normal keratinocytes co-cultured with normal fibroblasts, and (2) fibroblasts grown in serum-free media in the absence of keratinocytes in the upper chamber. Fibroblasts were indirectly quantified by 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay, with results confirmed by DNA content measurement, at days 1 and 5 after the co- culture initiation.Significantly, increased proliferation was seen in fibroblasts co-cultured with keloid keratinocytes, as compared with the normal keratinocyte controls at day 5 (analysis of variance, p < 0.001). These results strongly suggest that the overlying epidermal keratinocytes of the keloid may have an important, previously unappreciated role in keloid pathogenesis using paracrine or epithelial-mesenchymal signaling.     

The ratio of lipidperoxides to superoxide dismutase activity in the skin lesions of patients with severe skin diseases: an accurate prognostic indicator

We studied 35 patients with active inflammatory skin diseases, measuring the levels of lipidperoxides and of the oxygen radical scavenging enzyme superoxide dismutase (SOD) in biopsy specimens of skin lesions. Lipidperoxide levels were markedly elevated in all patients. In fifteen patients with disease that was severe and highly resistant to therapy, SOD activity was only slightly increased, in comparison with normal controls. In contrast, in the twenty patients with mild disease that responded well to therapy, SOD activity was markedly elevated. The ratio of lipidperoxide levels to SOD activity was thus an accurate prognostic indicator, being elevated only in the group not responding to treatment. These findings suggest that the severity of allergic inflammatory skin disease and/or the response to treatment may in part be governed by the degree to which the patient's SOD activity is up-regulated in response to the generation of tissue-damaging substances such as lipidperoxides. Interestingly, our studies revealed the SOD activities of both normal and inflamed skin to be unexpectedly high; our data suggest that SOD plays a critical role in protecting the skin from the effects of oxygen radicals and ultraviolet light.