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• W2094865794 abstract "Ceramides are the most abundant lipids constituting the intercellular matrix of the skin stratum corneum and their critical role in skin homeostasis has been extensively documented. Their concentration in the skin highly depends on the rate of availability of the enzymes involved in ceramide generation. The aim of this study was to investigate whether the concentration of prosaposin was altered in the skin of patients with psoriasis vulgaris. Prosaposin, the precursor of saposins (sphingolipid activator proteins), was measured in lesional and nonlesional skin of psoriatic patients and in normal skin from surgical patients, both at the mRNA and at the protein level. Densitometric analysis of reverse transcriptase–polymerase chain reaction bands separated by gel-electrophoresis showed a progressive decrease of prosaposin mRNA expression in nonlesional and lesional psoriatic skin, being substantially decreased in lesional psoriatic skin compared with normal control skin. Immunohistochemical analysis showed a significant decrease of prosaposin level in the stratum corneum of psoriatic lesional skin (both in active-type and in chronic-type plaque) compared with nonlesional and with normal skin (p <0.01), and in psoriatic nonlesional skin compared with normal control (p <0.05). Immunolocalization of sphingomyelinase in lesional and nonlesional psoriatic skin showed a decrease in the level of this enzyme in the stratum corneum of psoriatic lesional, compared with nonlesional skin. These results support the concept that disturbance of epidermal barrier function caused by derangement in ceramide generation can be crucial for the development of psoriatic skin diseases. Ceramides are the most abundant lipids constituting the intercellular matrix of the skin stratum corneum and their critical role in skin homeostasis has been extensively documented. Their concentration in the skin highly depends on the rate of availability of the enzymes involved in ceramide generation. The aim of this study was to investigate whether the concentration of prosaposin was altered in the skin of patients with psoriasis vulgaris. Prosaposin, the precursor of saposins (sphingolipid activator proteins), was measured in lesional and nonlesional skin of psoriatic patients and in normal skin from surgical patients, both at the mRNA and at the protein level. Densitometric analysis of reverse transcriptase–polymerase chain reaction bands separated by gel-electrophoresis showed a progressive decrease of prosaposin mRNA expression in nonlesional and lesional psoriatic skin, being substantially decreased in lesional psoriatic skin compared with normal control skin. Immunohistochemical analysis showed a significant decrease of prosaposin level in the stratum corneum of psoriatic lesional skin (both in active-type and in chronic-type plaque) compared with nonlesional and with normal skin (p <0.01), and in psoriatic nonlesional skin compared with normal control (p <0.05). Immunolocalization of sphingomyelinase in lesional and nonlesional psoriatic skin showed a decrease in the level of this enzyme in the stratum corneum of psoriatic lesional, compared with nonlesional skin. These results support the concept that disturbance of epidermal barrier function caused by derangement in ceramide generation can be crucial for the development of psoriatic skin diseases. prosaposin saposin sphingomyelin sphingomyelinase transepidermal water loss The intercellular space of the stratum corneum is an integral part of the epidermal permeability barrier and is packed with lamellar sheets of lipids as described in the popular ‘‘brick and mortar’' model (Elias, 1983Elias P.M. Epidermal lipids, barrier function, and desquamation.J Invest Dermatol. 1983; 80: 44-49Crossref PubMed Google Scholar). Stratum corneum lipids derive from exocytosis of lamellar granules into the intercellular space and are important factors preventing transepithelial permeation of water and of water soluble material (Landmann, 1988Landmann L. The epidermal permeability barrier.Anat Embryol. 1988; 178: 1-13Crossref PubMed Scopus (181) Google Scholar;Wertz, 1992Wertz P.W. Epidermal lipids.Semin Dermatol. 1992; 11: 106-113PubMed Google Scholar). They mainly consist of ceramides, and to a lesser extent of free cholesterol, cholesterol sulfate, and free fatty acids (Wertz et al., 1987Wertz P.W. Swartzendruber D.C. Abraham W. Madison K.C. Downing D.T. Essential fatty acids and epidermal integrity.Arch Dermatol. 1987; 123: 1381-1384Crossref PubMed Scopus (101) Google Scholar). Ceramides represent a unique, heterogeneous group of at least six different ceramides (CER 1–6) that differ from each other in the architecture of the polar head group and in the length of the nonpolar fatty acid chains (Wertz and Downing, 1983Wertz P.W. Downing D.T. Ceramides of pig epidermis: structure determination.J Lipid Res. 1983; 24: 759-765Abstract Full Text PDF PubMed Google Scholar;Wertz et al., 1985Wertz P.W. Miethke M.C. Long S.A. Strauss J.S. Downing D.T. The composition of the ceramides from human stratum corneum and from comedones.J Invest Dermatol. 1985; 84: 410-412Crossref PubMed Scopus (309) Google Scholar;Kerscher et al., 1991Kerscher M. Korting H.C. Schäfer-Korting M. Skin ceramides: structure and function.Eur J Dermatol. 1991; 1: 39-43Google Scholar). Ceramides have shown to be critical in epidermal permeability barrier homeostasis (Holleran et al., 1991Holleran W.M. Man M.Q. Gao W.N. Menon G.K. Elias P.M. Feingold K.R. Sphingolipids are required for mammalian epidermal barrier function. Inhibition of sphingolipid synthesis delays barrier recovery after acute perturbation.J Clin Invest. 1991; 88: 1338-1345Crossref PubMed Scopus (213) Google Scholar). There are two main pathways for ceramide generation. One is represented by the degradation of glucosyl-ceramides, catalyzed by β-glucocerebrosidase, and the other by the hydrolysis of sphingomyelin (SM) catalyzed by sphingomyelinase (SMase). Both the early activation of SMase and the later degradation of glucosyl-ceramides is essential for skin barrier repair (Holleran et al., 1994Holleran W.M. Takagi Y. Menon G.K. Jackson S.M. Lee J.M. Feingold K.R. Elias P.M. Permeability barrier requirements regulate epidermal β-glucocerebrosidase.J Lipid Res. 1994; 35: 905-912Abstract Full Text PDF PubMed Google Scholar;Jensen et al., 1999Jensen J.-M. Schütze S. Förl M. Krönke M. Proksch E. Roles for tumor necrosis factor receptor p55 and sphingomyelinase in reparing the cutaneous permeability barrier.J Clin Invest. 1999; 104: 1761-1770Crossref PubMed Scopus (164) Google Scholar). The lysosomal degradation of various glicosphingolipids also require the presence of nonenzymatic cofactors, the saposins (SAP) or so-called sphingolipid activator proteins, four of which (SAP A-D) derive from a common precursor, prosaposin (pSAP), by proteolytic cleavage and glycosylation. The structure, function, and distribution of SAP and of pSAP in mammalian tissues and body fluids have been thoroughly described (Sano et al., 1989Sano A. Hineno T. Mizuno T. Kondoh K. Ueno S. Kakimoto Y. Inui K. Sphingolipid hydrolase activator proteins and their precursors.Biochem Biophys Res Commun. 1989; 165: 1191-1197Crossref PubMed Scopus (70) Google Scholar;O'Brien and Kishimoto, 1991O'Brien J.S. Kishimoto Y. Saposin proteins: structure, function, and role in human lysosomal storage disorders.FASEB J. 1991; 5: 301-308Crossref PubMed Scopus (292) Google Scholar;Ito et al., 1993Ito K. Takahashi N. Takahashi A. Shimada I. Arata Y. O'Brien J.S. Kishimoto Y. Structural study of the oligosaccharide moieties of sphingolipid activator proteins, saposins A, C and D obtained from the spleen of a Gaucher patient.Eur J Biochem. 1993; 215: 171-179Crossref PubMed Scopus (19) Google Scholar;Hiraiwa et al., 1993Hiraiwa M. O'Brien J.S. Kishimoto Y. Galdzicka M. Fluharty A.L. Ginns E.I. Martin B.M. Isolation, characterization, and proteolysis of human prosaposin, the precursor of saposins (sphingolipid activator proteins).Arch Biochem Biophys. 1993; 304: 110-116Crossref PubMed Scopus (90) Google Scholar). SAP A-D, with molecular weight ranging from 12 to 14 kDa and with homologous structure, are specific activators of different hydrolases (Hiraiwa et al., 1993Hiraiwa M. O'Brien J.S. Kishimoto Y. Galdzicka M. Fluharty A.L. Ginns E.I. Martin B.M. Isolation, characterization, and proteolysis of human prosaposin, the precursor of saposins (sphingolipid activator proteins).Arch Biochem Biophys. 1993; 304: 110-116Crossref PubMed Scopus (90) Google Scholar). The activity of β-glucocerebrosidase has been shown to be stimulated by SAP-C (Wilkening et al., 1998Wilkening G. Linke T. Sandhoff K. Lysosomal degradation on vesicular membrane surfaces.J Biol Chem. 1998; 273: 30271-30278Crossref PubMed Scopus (136) Google Scholar), and the activity of acid SMase is stimulated by SAP-D (Morimoto et al., 1988Morimoto S. Martin B.M. Kishimoto Y. O'Brien J.S. Saposin D. A sphingomyelinase activator.Biochem Biophys Res Commun. 1988; 156: 403-410Crossref PubMed Scopus (99) Google Scholar;Tayama et al., 1993Tayama M. Soeda S. Kishimoto Y. Martin B.M. Callahan J.W. Hiraiwa M. O'Brien J.S. Effect of saposins on acid sphingomyelinase.Biochem J. 1993; 290: 401-404Crossref PubMed Scopus (27) Google Scholar). Although the major function of both pSAP and SAP consists in the lysosomal degradation of sphingolipids, they also play a role in transporting sphingolipids in the extracellular compartment and in different human secretory fluids (Hineno et al., 1991Hineno T. Sano A. Kondoh K. Ueno S. Kakimoto Y. Yoshida K. Secretion of sphingolipid hydrolase activator precursor, prosaposin.Biochem Biophys Res Comm. 1991; 176: 668-674Crossref PubMed Scopus (101) Google Scholar;Fürst and Sandhoff, 1992Fürst W. Sandhoff K. Activator proteins and topology of lysosomal sphingolipid catabolism.Biochem Biophys Acta. 1992; 1126: 1-16Crossref PubMed Scopus (248) Google Scholar). Decreased levels of pSAP have been detected in the skin of patients with atopic dermatitis, suggesting a role for pSAP in skin barrier disorders (Chang-Yi et al., 1997Chang-Yi C. Kusuda S. Seguchi T. Takahashi M. Aisu K. Tezuka T. Decreased level of prosaposin in atopic skin.J Invest Dermatol. 1997; 109: 319-323Abstract Full Text PDF PubMed Scopus (31) Google Scholar). The role of pSAP and of mature SAP in the epidermal permeability barrier formation has been recently described (Doering et al., 1999Doering T. Holleran W.M. Potratz A. Vielhaber G. Elias P.M. Suzuki K. Sandhoff K. Sphingolipid activator proteins are required for epidermal permeability barrier formation.J Biol Chem. 1999; 274: 11038-11045Crossref PubMed Scopus (135) Google Scholar). The authors showed that SAP-deficient mice are characterized by epidermal accumulation of glucosylceramides and SM paralleled by ceramide deficiency, by alteration of lipids binding to the cornified envelopes, by a thickened stratum lucidum with evidence of scaling, and, lastly, by abnormality in lamellar membrane maturation. Psoriasis is a benign inflammatory hyperproliferative disease, presenting an abnormal stratum corneum and a defective skin barrier function. Comparative studies with normal human skin have shown progressive increase in transepidermal water loss (TEWL) in the lesional skin of psoriatic patients with increasing severe phenotypes (Ghadially et al., 1996Ghadially R. Reed J.T. Elias P.M. Stratum corneum structure and function correlated with phenotypes in psoriasis.J Invest Dermatol. 1996; 107: 558-564Crossref PubMed Scopus (167) Google Scholar), despite a preservation of the electrolyte barrier (Grice et al., 1973Grice K. Sattar H. Baker H. The cutaneous barrier to salts and water in psoriasis and in normal skin.Br J Dermatol. 1973; 88: 459-463Crossref PubMed Scopus (36) Google Scholar). An ultrastructural analysis of the stratum corneum of psoriatic lesions has revealed extremely narrow intercellular spaces between a large number of parakeratotic corneocytes containing few pathologic epidermal lipids lamellae (Fartasch, 1997Fartasch M. Epidermal barrier in disorders of the skin.Microsc Res Tech. 1997; 38: 361-372Crossref PubMed Scopus (102) Google Scholar). These morphologic findings are consistent with biochemical studies that showed a decrease in relative free fatty acid content and a different pattern in ceramide distribution in psoriatic plaques, compared with normal control skin (Motta et al., 1993Motta S. Monti M. Sesana S. Caputo R. Carelli S. Ghidoni R. Ceramide composition of the psoriatic scale.Biochim Biophys Acta. 1993; 1182: 147-151Crossref PubMed Scopus (382) Google Scholar,Motta et al., 1994bMotta S. Sesana S. Monti M. Giuliani A. Caputo R. Interlamellar lipid differences between normal and psoriatic stratum corneum.Acta Derm Venereol (Stockh). 1994; 186: 131-132Google Scholar). In order to investigate whether the alterations in barrier function seen in psoriatic skin diseases could be related to disturbances in the pathways of ceramides generation, in this study we chose to focus on the activator protein system; we quantified both the reverse transcriptase–polymerase chain reaction (RT-PCR) products and the protein expression of pSAP in normal skin and in lesional and nonlesional psoriatic epidermis. Because pSAP expression seems to be correlated with epidermal SM content (Doering et al., 1999Doering T. Holleran W.M. Potratz A. Vielhaber G. Elias P.M. Suzuki K. Sandhoff K. Sphingolipid activator proteins are required for epidermal permeability barrier formation.J Biol Chem. 1999; 274: 11038-11045Crossref PubMed Scopus (135) Google Scholar), and because acid SMase has been shown to be important in skin permeability barrier repair (Jensen et al., 1999Jensen J.-M. Schütze S. Förl M. Krönke M. Proksch E. Roles for tumor necrosis factor receptor p55 and sphingomyelinase in reparing the cutaneous permeability barrier.J Clin Invest. 1999; 104: 1761-1770Crossref PubMed Scopus (164) Google Scholar), we furthermore localized acid SMase by immunohistochemistry and compared its protein expression in lesional and nonlesional psoriatic skin. Fresh skin tissue biopsies were obtained from the lesional skin of patients with psoriasis vulgaris (n = 8, two females and six males, with age ranging from 30 to 68 y, with a mean of 54 y). Three male patients among the aforementioned group (age ranging from 30 to 45 y, with a mean of 37 y) donated both lesional and nonlesional skin. Normal control skin (n = 8) was obtained from uninvolved areas of surgical tumor resections (basalioma, keratoma). All specimen underwent a histopathologic examination after routine hematoxylin and eosin (H&E) staining and the additional immunohistochemical detection of phospholipase A2, type II, was used to grade the severity of the psoriatic lesions (Ragaz and Ackerman, 1979Ragaz A. Ackerman A.B. Evolution, maturation, and regression of lesions of psoriasis.Am J Dermatopathol. 1979; 1: 199-214Crossref PubMed Scopus (156) Google Scholar;Andersen et al., 1994Andersen S. Sjursen W. Laegreid A. Volden G. Johansen B. Elevated expression of human nonpancreatic phospholipase A2 in psoriatic tissue.Inflammation. 1994; 18: 1-12Crossref PubMed Scopus (69) Google Scholar). The histopathologic examination of the psoriatic skin tissue sections stained with H&E followed by the localization of PLA2, type II, revealed that four of them were typical examples of ‘‘active plaque’' psoriasis and four of them of ‘‘chronic plaque’' psoriasis. ‘‘Active plaque’' psoriasis was characterized by a greater infiltration of inflammatory cells and a higher number of PLA2, type II-positive fibroblasts in the dermis. All chemicals were purchased from Sigma (Deisenhofen, Germany), unless otherwise specified. The mRNA was isolated from the specimens obtained from both lesional and nonlesional areas of the three male patients mentioned above with ‘‘active plaque’' psoriasis vulgaris, and from one normal control subject. Three additional samples were obtained from the same patients by simply scraping off the psoriatic lesions. For RNA isolation, the biopsies were washed in phosphate buffered saline (PBS) and digested in 0.5% dispase solution for 2 h at 37°C. Then the epidermis was mechanically separated, frozen in liquid nitrogen, and stored at -70°C. The epidermis was harvested in solution buffer (supplied by QuickPrep® mRNA, Pharmacia, Uppsala, Sweden) and sonicated for 10 s to destroy the cell membranes without degrading the nucleic acids. Further isolation was performed in three steps following the suppliers protocol (Pharmacia). After ethanol precipitation the harvested mRNA was diluted in 50 μl H2O aliquots. For reverse transcription mRNA probes were diluted in 11 μl H2O and heated together with 0.5 μg oligo (dt)12-18-primer at 70°C for 10 min. The RT-reaction was performed at 42°C in 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl2, 0.01 M DTT, 1 mM dNTP mix and 200 U SUPERSCRIPT II RT (Gibco BRL, Eggenstein, Germany) for 60 min and stopped at 70°C for 15 min. To nick the mRNA-strand, the sample was incubated with 2 IU RNAse H (Gibco BRL) for 20 min at 37°C. For each experiment, primer concentrations, RNA amounts, and PCR cycles were titrated to establish standard conditions. The validity of quantitative comparisons of PCR products was insured by using a number of PCR cycles mapped to, within the linear curve. For PCR, 1 μl to 3 μl of the sample was incubated in 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTP mix, and 0.2 pmol of pSAP and GAPDH primers (see below). The thermal profile used on a Hybaid Limited TouchDown Thermal Cycler (Hybaid, Middlesex, U.K.) consisted on denaturation at 94°C for 45 s, annealing at 53°C for 30 s, and an extension temperature of 72°C for 1 min for 32 cycles. By optimized conditions, the PCR runs were repeated on the same RNA samples to assure the reproducibility of the data. The oligonucleotides used were chosen overlapping introns, to avoid the amplification of genomic DNA. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control. GAPDH sense, 5′-GGTGAAGGTCGGAGTCAACGGA-3′ GAPDH antisense, 5′-GAGGGATCTCGCTCCTGGAAGA-3′ pSAP sense, 5′-GCCTGCACTGACCGTTCACGTG-3′ pSAP antisense, 5′-GGCTCCAATTTTCAAGCACACG-3′. PCR reactions were separated by gel electrophoresis on 2% agarose gels stained with 0.4 mg ethidium bromide per ml. The gels were visualized over an on-line UV light source transilluminator (Gel Doc 1000 Video documentation system, Bio-Rad, Hercules, CA). The PCR products of the expected size (265 bp for prosaposin and 430 bp for GAPDH) were then manually defined and the bands intensity was quantified using Quantity One quantitation software (Bio-Rad, Hercules, CA). The intensity of the PCR bands of pSAP were expressed normalized to GAPDH. For the immunohistochemical study, all biopsies of psoriatic lesional, nonlesional, and of normal control skin were snap frozen in liquid nitrogen for cryostat sectioning. Frozen tissue samples were submerged in Optimum Cutting Temperature embedding compound (OCT, Tissue-Tek, Miles Laboratory, Elkhart, IN) and cryostat sections (6 μm) were prepared on positively charged slides and allowed to air-dry. Tissue fixation was obtained with fresh 2% paraformaldehyde in phosphate-buffered saline (PBS), for 5 min, and methanol, for 10 min at 4°C. Part of the biopsies were fixed by immersion in 3.7% formaldehyde in PBS at 4°C for 4 h, and embedded in paraffin, using an automatic apparatus (Tissue-TEK VIP, Miles Scientific). The melted wax temperature did not exceed 56°C. Fixed and embedded tissue blocks were allowed to cool and were stored in plastic cassettes, ready to be cut. The sections were prepared on twice-washed polylysine-coated slides, and deparaffinized. All tissue sections were further processed for indirect immunohistochemistry with an ABC-based method (Vector Laboratories, Burlingame, CA). After blocking both endogenous biotin binding sites with sequential incubations of avidin and biotin, and endogenous peroxidases with 15 min incubation in methanol/1% H2O2, the sections were subjected to overnight incubation at 4°C with primary specific antibodies anti-SAP-D, antiphospholipase A2, type II, or control solutions. Tissue sections obtained from the involved and uninvolved psoriatic skin of three patients were additionally stained with primary specific antibody anti-SMase. Rabbit polyclonal antibodies against SAP-D and human acid SMase were generously donated by Dr. T. Tezuka, Department of Dermatology of the Kinki University School of Medicine, Osaka, Japan (Chang-Yi et al., 1997Chang-Yi C. Kusuda S. Seguchi T. Takahashi M. Aisu K. Tezuka T. Decreased level of prosaposin in atopic skin.J Invest Dermatol. 1997; 109: 319-323Abstract Full Text PDF PubMed Scopus (31) Google Scholar). The antiserum was diluted 1:1000 and 1:2000 for anti-SAP-D and for anti-SMase, respectively. Monoclonal antibodies against phospholipase A2, type II, were purchased from Boehringer Mannheim (Mannheim, Germany). The working concentration of 10 μg per ml showed best results. As a positive control, a monoclonal anticytokeratin PAN antibody (Boehringer Mannheim) was used at a concentration of 10 μg per ml. Negative controls included use of buffer alone or dilutions of nonspecific purified rabbit and mouse IgG in the primary layer (Vector Laboratories, Burlingame, CA). Antibodies were diluted in 0.1% bovine serum albumin (BSA) and 0.1% sodium azide (Roth, Karlsruhe, Germany) in PBS (pH 7.4). Specific binding was detected using a biotin-conjugated horse antimouse and antirabbit IgG, avidin-biotin peroxidase complex (Vector Laboratories), and a substrate solution of H2O2 (0.03%) and diaminobenzidine (2 mg per ml) in 0.5 M Tris-saline, pH 7.6, with 1 M imidazole (Roth, Karlsruhe, Germany) and 0.3% sodium azide (Roth). The slides were then counterstained with hematoxylin, dehydrated through graded ethanols and xylene, mounted, and coverslipped. A densitometric analysis of the stratum corneum immunoreactivity with antisaposin-D of all high quality samples, in which the stratum corneum was not damaged during tissue preparation procedures [psoriatic lesional (n = 7), nonlesional (n = 3), and normal control skin (n = 8)] was used as an alternative of the widely used scoring method. The analysis was performed in a blinded fashion. Three images from each tissue section (together covering about 80%-90% of the tissue section) were randomly selected, recorded with a camera, and converted to digital files (Overlay Framegrubber, Imaging Technology, Wobuln, MA). The camera (CCD-Kamera VS 450, Stemmer Imaging, Puchheim, Germany) was within 10% equally sensitive to all wavelengths in the whole visible spectrum, where the absorption peak of oxidized diaminobenzidine, used as substrate in the immunohistochemical study, is 465 nm (Fahimi and Herzog, 1973Fahimi H.D. Herzog V. A colorimetric method for measurement of the (peroxidase-mediated) oxidation of 3,3′-diaminobenzidine.J Histochem Cytochem. 1973; 21: 499-502Crossref PubMed Scopus (35) Google Scholar). Three densitometric measurements normalized to identical surface area of the stratum corneum were performed per each recorded image using Optimas 6 software (Optimas Corporation, Bothell, Washington; http://www.optimas.com). This software analyses digitized gray values and provides a quantitative measurement of the immunoreactivity in terms of active pixels in the selected areas, and the number recorded represents the mean density enclosed by the selected areas, where white is 0 and black is 255. To quantify the interference of the counterstained parakeratotic nuclei, a densitometric analysis on parallel sections stained with nonspecific antibody (negative control) and counterstained has been performed. For statistical evaluation, one-way analysis of variance (ANOVA) with repeated measures and posthoc pairwise comparisons (Scheffé test) were used. Data were expressed as mean ±SEM. A p value below 0.05 was considered to be significant. The separation of pSAP and GAPDH RT-PCR products by gel electrophoresis and ethidium bromide staining showed a progressive decrease in the expression of pSAP mRNA in the nonlesional (a) and lesional (b) skin of psoriatic patients (P1, P2, P3), compared with control (C) (Figure 1). The semiquantitative assessment of the RT-PCR bands are represented normalized to GAPDH expression (Figure 2). Obvious is the progressive decrease of pSAP expression in the nonlesional and in the lesional skin of psoriatic patients compared with control. The densitometric values of pSAP-expression in lesional psoriatic skin are consistently lower compared with control.Figure 2Semiquantitative analysis of pSAP PCR bands. Relative density levels of pSAP PCR bands are expressed in percentage of GAPDH. C = Normal control skin; P1, P2, P3 = three psoriatic patients, uninvolved skin left, involved skin right. Densitometric values obtained from the corresponding ethidium bromide-stained PCR reaction products in Figure 1 are presented.View Large Image Figure ViewerDownload (PPT) The three samples obtained by simply scraping off the psoriatic lesions resulted in nonsignificant PCR bands (data not shown). All samples investigated showed a very similar pattern of immunoreactivity, therefore representative samples are shown (Figure 3, Figure 4 and Figure 5). In normal control skin, pSAP-like protein was localized as spotty reactions throughout all the epidermal layers, but most abundantly in the granular-corneocyte transition zone and in the stratum corneum (arrow). Also dermal fibroblasts, mast cells, and mononuclear cells stained intensively (Figure 3a). The negative control showed no stain (Figure 3b). In psoriatic nonlesional skin, the immunolocalization of pSAP-like protein was similar to control skin, and it was present both in the granular-corneocyte transition zone and in the stratum corneum (arrows), although it was less abundant (Figure 4a). In lesional psoriatic skin, spotty reactions of pSAP-like protein were still immunohistochemically detectable at the granular-corneocyte transition zone (arrow), but a marked decrease of pSAP-like protein was evident in the stratum corneum (Figure 4b). There was no difference in the level of pSAP-like protein between the patients with ‘‘active type’' and ‘‘chronic type’' psoriasis. Dermal inflammatory cells and fibroblasts maintained their labeling with anti-SAP-D (Figure 4b, small arrow). Quantification of pSAP-like immunoreactivity by densitometric analysis of the stratum corneum of normal control skin, psoriatic nonlesional, and psoriatic lesional skin showed that in psoriatic lesional skin the level of pSAP-like protein was significantly lower compared with control and with psoriatic nonlesional skin (p <0.01, Table 1). The data furthermore indicated that the level of pSAP-like protein is decreased also in the stratum corneum of psoriatic nonlesional skin with respect to the normal control group (p <0.05, Table 1). The intersubject variation of the psoriatic nonlesional group was higher compared with the other samples, although all three samples investigated in this group showed lower expression of pSAP-like protein in the stratum corneum compared with normal control skin, and higher expression compared with psoriatic lesional skin. The densitometric analysis of the stratum corneum of psoriatic lesional skin stained with nonspecific primary antibody and counterstained with hematoxylin showed no statistical difference to the parallel sections stained with SAP-D antibody (Table 1). This result demonstrates the low interference of the parakeratotic nuclei counterstain in the densitometric evaluation.Figure 4Immunohistochemical localization of pSAP-like protein in psoriatic epidermis. In nonlesional psoriatic epidermis (a) pSAP-like protein is visible in the granular-corneocyte transition zone and in the lower stratum corneum (arrows). In lesional psoriatic epidermis (b) pSAP-like protein is decreased in whole epidermis, but especially in the stratum corneum. Spotty reaction are confined to the granular-corneocyte transition zone (arrow). Positive staining of an inflammatory cell in the dermis is also evident (small arrow). Scale bar: 30 μm.View Large Image Figure ViewerDownload (PPT)Figure 5Immunohistochemical localization of SMase in psoriatic epidermis. SMase was immunohistochemically detected in tissue sections of nonlesional and lesional psoriatic skin using the ABC method (see Materials and Methods). In nonlesional psoriatic skin (a) SMase can be visualized in the periphery of and weakly within the cells of stratum spinosum, stratum granulosum, lucidum, and stratum corneum (arrow), being absent in the stratum basale (small arrow). In lesional psoriatic epidermis (b) SMase is localized as dotty reactions in the granular-corneocyte transition zone (arrow). Scale bar: 30 μm.View Large Image Figure ViewerDownload (PPT)Table IDensitometric analysis of skin stratum corneum following immunostaining with antisaposin D and with nonspecific IgG primary antibodiesSkin samplesncEach n represents the number of skin samples assessed. Each sample was represented by nine measurements (see Materials and Methods).MeandA densitometric analysis of the stratum corneum was performed using Optimas 6 software. The number represents the mean density enclosed by the area of interest, where white is 0 and black is 255.SEMNormal controlaTissue samples were analysed following immunostaining either with anti saposin-D antibody (a) or with nonspecific rabbit IgG (negative control, b) using the ABC method, then counterstained with hematoxyl" @default.
• W2094865794 created "2016-06-24" @default.
• W2094865794 creator A5014484985 @default.
• W2094865794 creator A5015015906 @default.
• W2094865794 creator A5030745516 @default.
• W2094865794 creator A5062117902 @default.
• W2094865794 date "2001-03-01" @default.
• W2094865794 modified "2023-10-11" @default.
• W2094865794 title "The Level of Prosaposin is Decreased in the Skin of Patients with Psoriasis Vulgaris" @default.
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