Invasion of Candida albicans Correlates with Expression of Secreted Aspartic Proteinases during Experimental Infection of Human Epidermis

Invasion of Candida albicans Correlates with Expression of Secreted Aspartic Proteinases during Experimental Infection of Human Epidermis Martin Schaller, Carolin Schackert, Hans C. Korting, Elfriede Januschke, and Bernhard Hube*

Department of Dermatology, Ludwig-Maximilians-University, Munich, Germany; *Institute for General Botany, Applied Molecular Biology III, University of Hamburg, Germany

Secreted aspartic proteinases (Saps) encoded by 10 genes of Candida albicans are important virulence factors for different types of candidiasis. Distinct SAP genes have previously been shown to contribute to tissue damage in a model of oral candidiasis. In this study a progressive SAP expression in the order SAP1 and SAP2 > SAP8 > SAP6 > SAP3 was observed in an in vitro model of cutaneous candidiasis based on reconstituted human epidermis. Transcripts of SAP1 and SAP2 were detected during initial invasion of the stratum corneum by C. albicans. Deeper, extensive penetration of the corneal layer was accompanied by additional SAP8 mRNA. SAP6

expression occurred concomitantly with germ tube formation and extensive hyphal growth in the strata granulosum, spinosum, and basale. Ultrastructural studies using speci®c polyclonal antibodies directed against the gene products of SAP1±3 and SAP4±6 revealed predominant expression of Sap1±3. The protective effect of the aspartic proteinase inhibitor pepstatin A during infection of the epidermis and an attenuated virulence phenotype of SAP-de®cient mutants suggest that the observed SAP expression correlates with tissue damage in the skin. Key words: cutaneous candidiasis/immunoelectron microscopy/ RT-PCR. J Invest Dermatol 114:712±717, 2000

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Interaction of C. albicans with murine macrophages revealed the expression of the closely related genes SAP4±6 (Borg-von Zepelin et al, 1998). The Dsap4±6 mutant was killed 53% more effectively after contact with macrophages than the parental strain (Borg-von Zepelin et al, 1998). In addition, an important role for SAP4±6 but not for SAP1±3 was observed in a mouse peritonitis model (Kretschmar et al, 1999). These results support a role of SAP4±6 in pathogenicity of invasive candidiasis. In contrast, other data suggest that the isoenzymes Sap1±3 are essential for mucosal infections. Attenuated virulence of the Dsap1, Dsap2, and Dsap3 but not Dsap4±6 mutants in comparison with the parental strain was demonstrated during experimental vaginal and oral candidiasis (De Bernardis et al, 1999; Schaller et al, 1999c). In normal patients, cutaneous candidiasis is occasionally observed and is restricted to areas of skin folds. In immunocompromised patients, however, skin infections with C. albicans are common diseases. In some of these patients extensive cutaneous candidiasis may even lead to a serious, life-threatening, systemic infection (Odds, 1988). In this study we investigated histologic alterations of human skin caused by C. albicans infections. The expression pattern of SAP genes and the ultrastructural localization of distinct groups of Sap antigens were monitored during the infection process and the relevance of expressed Saps was demonstrated using a speci®c aspartic proteinase inhibitor.

ecretion of hydrolytic enzymes such as aspartic proteinases by Candida albicans has often been considered as pathogenic factor (Cutler, 1991; Odds, 1994). To date, gene sequences of 10 different secreted aspartic proteinases (SAP1±10) have been reported (Monod et al, 1994, 1998; Hube, 1996; Felk, SchaÈfer, Hube, unpublished data). Expression studies have demonstrated transcripts for SAP1 and SAP2 in a rat vaginitis model by northern analysis (De Bernardis et al, 1995) and a temporal progression of SAP gene expression in the order SAP1 and SAP3 > SAP6 > SAP2 and SAP8 in the in vitro model of oral candidiasis by reverse transcriptase polymerase chain reaction (Schaller et al, 1998). In vivo analysis of samples from patients with oral candidiasis and asymptomatic carriers demonstrated variable patterns of SAP gene expression (Schaller et al, 1998; Naglik et al, 1999). Transcripts for SAP1 and SAP3 were predominantly found in oral candidiasis patients but not in asymptomatic carriers (Naglik et al, 1999). Generation of different SAP null mutants (Hube et al, 1997; Sanglard et al, 1997; Schaller et al, 1999c) provides a tool to clarify the relevance of distinct Saps for their pathogenicity in model systems of candidiasis. For disseminated infections an attenuated virulence has been demonstrated by a moderate increase in survival rate of mice and guinea pigs after intravenous infection with Dsap1, Dsap2, Dsap3 (Hube et al, 1997) or Dsap4±6 (Sanglard et al, 1997). Manuscript received October 7, 1999; revised December 9, 1999; accepted for publication December 19, 1999. Reprint requests to: Dr M. Schaller, Department of Dermatology, Ludwig-Maximilians-UniversitaÈt, Frauenlobstr. 9±11, D-80337 MuÈnchen, Germany. Abbreviations: SAP, secreted aspartic proteinase (gene); Sap, secreted aspartic proteinase (protein). 0022-202X/00/$15.00

MATERIALS AND METHODS Candida strains, culture media, and growth conditions For this study the clinical C. albicans isolate designated SC5314 (Gillum et al, 1984) and the SAP null mutants Dsap1 (BH24±15±1) and Dsap2 (BH52±1-17) were used (Hube et al, 1997). For the infection of the reconstituted epidermis, inocula were prepared by culturing yeast cells for 24 h at 37°C

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on Sabouraud-dextrose-agar (Difco, Detroit, MI). A sample of the culture was washed three times in 0.9% NaCl. Approximately 2 3 105 cells were then suspended in 10 ml YPG medium (1% yeast extract, 2% peptone, and 2% glucose) (Difco). The suspension was cultured for 16 h at 25°C with orbital shaking. A suspension of 4 3 106 cells was incubated with shaking in fresh medium for 24 h at 37°C. After washing three times with phosphatebuffered saline (PBS), the ®nal inoculum was then adjusted to the desired density with PBS solution. Model of cutaneous candidiasis The reconstituted human epidermis for the in vitro model of cutaneous candidiasis was supplied by Skinethic Laboratory (Nice, France). Human keratinocytes derived from juvenile foreskins obtained during surgery were cultured on an inert supporting membrane (Rosdy and Claus, 1990; Rosdy et al, 1993). Cultures were incubated in serum-free conditions in a de®ned medium based on the MCDB 153 medium (Clonetics, San Diego, CA) containing 5 mg insulin per ml, on a 0.6 cm2 microporous polycarbonate ®lter for 14 d. The skin equivalent and all culture media were prepared without antibiotics and antimycotics. Three replicate infection experiments were performed with the C. albicans strain SC5314. The epidermis was infected with 2 3 106 C. albicans yeast cells in 100 ml PBS. Controls contained 100 ml PBS alone. In the ®rst set of experiments infected and uninfected cultures were incubated at 37°C with 5% CO2 at 100% humidity for 24, 36, 48, 96, 144, and 168 h. To con®rm the SAP expression pattern of these experiments two additional experimental infections were performed with incubation periods of 12 h, 24 h, and 48 h. Immunoelectron microscopy studies for detection of Sap antigen were carried out in infected samples taken after 24 and 48 h. Incubation periods for the pepstatin A experiments were 24 and 48 h. The medium was changed every 24 h. Infection experiments with the SAP null mutants Dsap1 and Dsap2 with incubation periods of 24 h and 48 h were also performed in triplicate as described above for SC5314. The growth rates of the SAP null mutants (Dsap1, Dsap2) and the SC5314 with and without application of pepstatin A were tested by cell counting 24 h and 48 h after infection of the epidermis. Epidermal pieces were suspended in 1 ml of PBS and vortexed for 10 min. The cell suspension was diluted 1:100 and the number of cells was determined with a Neubauer cell chamber.

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Pepstatin A experiments For inhibition of Saps, pepstatin A (Sigma) was dissolved in absolute methanol to give a stock solution of 100 mM and administered to 100 ml PBS containing 2 3 106 C. albicans yeast cells of the SC5314 strain at a ®nal concentration of 10 and 15 mM. This solution was used to infect the epidermis as described above. Controls contained 100 ml PBS with 10 and 15 mM pepstatin A alone. Incubation periods were 24 and 48 h. Light microscopy Light microscopy studies were performed as previously described (Schaller et al, 1998, 1999c) to evaluate histologic changes during infection. A part of each specimen was ®xed, post®xed, and embedded in glycide ether. The small blocks of tissue were cut using an ultra-microtome (Ultracut, Reichert, Vienna, Austria). Semi-thin sections (1 mm) were studied with a light microscope after staining with 1% toluidine blue and 1% pyronine G (Merck, Darmstadt, Germany). The histologic changes of the skin were evaluated on the basis of 50 sections from ®ve different sites for each infected epidermis. Immunoelectron microscopy Postembedding immunogold labeling was carried out as previously described (Schaller et al, 1999c) for intracellular detection of Sap antigen in SC5314 infected samples taken after 24 h and 48 h. After ®xation specimens were embedded in LR-White. Sections, 80±100 nm thick, were mounted on nickel grids. Grids were then incubated with anti-Sap polyclonal rabbit antibodies, directed against Sap1±3 or Sap4±6 (Borg-von Zepelin et al, 1998). After washing over night with PBS, grids were incubated with 10 nm gold-conjugated goat antirabbit IgG (Auroprobe EM Immunogold reagents, Amersham, U.K.). Grids were then ®xed with 2% glutaraldehyde and stained with 0.5% uranyl acetate for 10 min, and 2.7% lead citrate for 5 min (Ultrastainer, LKB, Sweden) at 20°C. For examination a Zeiss EM 902 transmission electron microscope (Zeiss, Oberkochen, Germany) was used, operating at 80 kV, at magni®cations between 30003 and 85,0003. RNA isolation, cDNA synthesis (reverse transcriptase), polymerase chain reaction, and pairs of primers Exact descriptions about RNA preparation, cDNA synthesis, primer sequences, and polymerase chain reaction conditions have been published previously (Schaller et al, 1998, 1999c).

Figure 1. Light micrographs of reconstituted human epidermis before, 24 h and 48 h after infection with C. albicans (SC5314). Strati®ed keratinocytes with stratum corneum (A). Regular distribution of C. albicans cells within the epidermis after 24 h. Hyperkeratosis of the stratum corneum with a parakeratotic cell (arrowhead). Multiple vacuoles within the cytoplasm, enlarged intercellular spaces (stars), and dyskeratotic cells (arrows) (B). Focal invasion of C. albicans in the epidermis with strong edema and detachment of the affected hyperkeratotic stratum corneum 24 h after infection. Vacuolization (v) and edema of the keratinocytes (C). Extensive invasion of C. albicans in the epidermis with edema and vacuolization in all keratinocyte layers 48 h after infection. Note the yeast to hyphal transition within the living epidermis (D). Scale bars: 20 mm.

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RESULTS Morphology of uninfected reconstituted human epidermis The uninfected tissue consisted of well-strati®ed keratinocytes with stratum corneum and showed the characteristic differentiation pattern of the epidermis (Fig 1A). Incubation of the epidermis with PBS for up to 168 h led to an increased number of the horny cell layers. Morphology of the epidermis after infection with C. albicans (SC5314) Histologic examination of multiple samples 24 h after challenge revealed two different patterns of epidermal infection by C. albicans (Fig 1B, C). Yeast cells could be observed within the hyperkeratotic stratum corneum with either a regular distribution (Fig 1B) or with focal invasion (Fig 1C) into the epidermis. The ®rst infection pattern was characterized by several C. albicans cells regularly distributed throughout the hyperkeratotic and parakeratotic stratum corneum. The corneocytes showed only slight edema, especially of the uppermost layer, and within the strata granulosum and spinosum only a few hyphal cells could be observed. Within the cytoplasm of the keratinocytes multiple vacuoles were visible and the intercellular spaces were enlarged as a sign of spongiosis. In the basal cell layer dyskeratotic cells were visible (Fig 1B). Histologic sections of the second pattern demonstrated an extensive invasion of C. albicans cells within a distinct and restricted area of the hyperkeratotic and parakeratotic stratum corneum. The corneocytes showed strong intracellular edema and a marked decrease in the horny layer coherence. Morphologic alterations of the epidermis were the same as described above but were enhanced (Fig 1C). Extensive penetration of C. albicans cells correlated with an increased number of hyphal elements, especially when the strata granulosum, spinosum, and basale were invaded. Subepidermal invasion led to detachment of the epidermis from the polycarbonate ®lter and was accompanied by hyphal to yeast transition. The increasing appearance of hyphal elements within the epidermis 36 h after infection coincided with marked edema of the keratinocytes. In the following 12 h C. albicans induced alterations such as detachment of stratum corneum cells, and marked edema of the keratinocytes could be discovered in all histologic sections. Again hyphal ®laments of C. albicans were mainly seen within the strata granulosum, spinosum, and basale, whereas yeast cells were located subepidermally and in the stratum corneum (Fig 1D). Acantholysis and edema increased in the later stages of infection (96±168 h). Further experiments were performed to illustrate the early steps of infection. Initial penetration of the stratum corneum with involvement of the uppermost corneocytes was demonstrated 12 h after infection. Histologic investigations 12 and 36 h later con®rmed the morphologic alterations demonstrated in Fig 1(B-D). In the infected samples hyperkeratosis (Fig 1B-D) of the stratum corneum, development of parakeratotic (Fig 1B) and dyskeratotic cells (Fig 1B), and spongiotic edema (Fig 1B-D) were characteristic features of the epidermis. Thus, several characteristic histologic features of human cutaneous candidiasis could be demonstrated (Sohnle and Kirkpatrick, 1978; Odds, 1988; Sohnle and Hahn, 1992; Luna and Tortoledo, 1993). SAP expression by C. albicans (SC5314) during the course of infection To evaluate whether the structural alterations of the epidermis during infection with C. albicans were due to proteolytic activity of the fungus, we investigated the expression of genes encoding secreted aspartic proteinases (SAPs) over the time course of infection. Total RNA was isolated from uninfected epidermis and from samples at different times during the infection process. No mRNA transcript was ampli®ed from the RNA of the uninfected epidermis by reverse transcriptase polymerase chain reaction (RTPCR) for either the SAP-speci®c primers or primers speci®c for the constitutively expressed gene encoding the elongation factor (EFB1) (Maneu et al, 1996). In all infected samples investigated, RT-PCR fragments 526 bp in size were ampli®ed with EFB1-

Figure 2. Analysis of RT-PCR products of RNA (lanes 2±8) from samples taken 24 h, 36 h, and 48 h after infection with C. albicans (SC5314). A 526 bp fragment size obtained by ampli®cation with EFB1 primers demonstrates the cDNA origin of the template (lane 9), whereas the same set of primers ampli®ed an 891 bp intron-containing fragment when genomic DNA was used as template (lane 10). In lane 1 the molecular mass marker pBR322 DNA/MvaI (M) (MBI Fermentas, St Leon-Rot, Germany) was used, giving fragments 1857, 1058, 929, and 383 bp in size.

speci®c primers, indicating the cDNA origin of the templates (Fig 2A-C). In the ®rst set of experiments SAP-gene-speci®c fragments were detected 24, 36, 48, 96, 144, and 168 h after infection. SAP1, SAP2, and SAP8 expression occurred after 24 h (Fig 2A), corresponding to the morphologic alterations demonstrated in Fig 1(B). Additional SAP6 transcripts were observed 12 h later (Fig 2B) and SAP3 expression occurred after 48 h (Fig 2C). This expression pattern was stable for the remaining period of investigation up to 168 h. In the second and third sets of infection experiments we also investigated samples of the early stages of infection when ®rst lesions with invasion of the uppermost corneocytes were visible. This stage of infection was accompanied by the expression of SAP1 and SAP2. Con®rming the RT-PCR results described above, transcripts for SAP1, SAP2, SAP8, and SAP1±3, SAP6, SAP8 were detected after 24 and 48 h.

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Figure 3. Electron microscopy with postembedding immunogold labeling, using 10 nm gold particles in samples taken 24 h after infection with C. albicans (SC5314). Sap1±3 antigen within the cell wall of a C. albicans yeast found on top of the epidermis without direct contact with the super®cial corneocytes (A). Deposition of Sap1±3 antigen was discovered within the cell wall of C. albicans yeast cells in direct contact with super®cial corneocytes (B) or within epidermal cells (C). Very few or no gold particles were found within the fungal cell wall of yeast cells at the stage of epithelial adhesion or invasion after labeling with the Sap4±6 speci®c antibody (D). Scale bars: 0.5 mm.

RT-PCR performed in the absence of reverse transcriptase showed no SAP signals, verifying the absence of genomic DNA contamination. Immunoelectron microscopy As expression of SAP genes correlated with lesions of the in vitro model, we used immunoelectron microscopy to demonstrate the presence of Sap antigens and to study the distribution of the proteinases in the epidermis. To distinguish the ultrastructural localization of different Saps in the in vitro model, we used two previously described polyclonal speci®c antibodies directed against Sap1±3 or Sap4±6 (Borg-von Zepelin et al, 1998). For detection of Sap immunoreactivity within C. albicans cells or keratinocytes, postembedding labeling with Sap antibodies conjugated to 10 nm gold particles was carried out 24 h and 48 h after infection with SC5314. RT-PCR analysis showed SAP1-SAP3, SAP6, and SAP8 transcripts in both these samples. Labeling with the Sap1±3 antibody demonstrated intensive density of Sap immunoreactivity in all C. albicans cells, including those found on top of the epidermis without direct contact to the corneocytes (Fig 3A), adherent to corneocytes (Fig 3B), or within epidermal cells (Fig 3C). Sap immunoreactivity was mainly localized next to the cell wall and the cytoplasmic membrane of C. albicans cells (Fig 3A-C). Labeling with the Sap antibody directed against Sap4±6 revealed only few gold particles in C. albicans yeast and hyphal elements (Fig 3D). No

speci®c gold labeling was seen in control experiments without the addition of the polyclonal antibodies Sap1±3 or Sap4±6. Infection experiments in the presence of pepstatin A We repeated the infection experiments in the presence or absence of the aspartic proteinase inhibitor pepstatin A to determine if the tissue lesion in the in vitro model was caused by Sap activity. In the absence of pepstatin A histologic changes after 24 and 48 h were similar to those demonstrated in Fig 1(B-D). These morphologic alterations were strongly decreased when pepstatin A was added at concentrations of 10 mM (Fig 4A) or 15 mM. In the presence of the inhibitor edema and detachment were observed mainly in the uppermost corneocyte layers of the stratum corneum. Edema and vacuolization of the keratinocytes of the strata granulosum, spinosum, and basale were strongly reduced and invasion of the epidermis by C. albicans was completely blocked by pepstatin A (Fig 4A). Incubation of reconstituted epidermis with pepstatin A alone at concentrations of 10 mM and 15 mM demonstrated no histologic alterations. To examine whether application of pepstatin A affected the growth rate during infection, growth was measured by counting cells 24 h and 48 h after infection of epidermis. Growth rates of the parental strain in untreated epidermis were identical to that after application of pepstatin A. Infection experiments with SAP null mutants As pepstatin A inhibited tissue damage, and SAP1 and SAP2 were the ®rst

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Figure 4. Light micrographs of reconstituted human epidermis 48 h after infection with C. albicans (SC5314) in the presence of 10 mM pepstatin and 48 h after infection with C. albicans Dsap1 and Dsap2. Adhesion of Candida cells to the super®cial corneocytes is seen, but no marked morphologic alterations are visible in the presence of pepstatin A. Invasion of the epidermis is completely blocked (A). Attenuated virulence phenotype of both mutants (Dsup1 (B), Dsup2 (C)) compared with the parental strain infection (Fig 3). Only the uppermost corneocytes show signs of keratinolytic degradation. Moderate vacuolization and spongiosis. Scale bars: 20 mm.

transcripts detected during the epidermal infection, we concluded that these genes must be important for the initial lesions. We therefore investigated the histologic phenotype of infections with Dsap1 and Dsap2 mutants. Both mutants showed markedly attenuated tissue damage (Fig 4B, C) compared with the infection with the parental strain SC5314 (histologic changes similar to Fig 1D). Histologic alterations were not completely blocked but keratinolytic degradation of the stratum corneum, spongiosis of the basal cell layer, and vacuolization were reduced (Fig 4B, C). DISCUSSION A model based on reconstituted human epidermis was used in this study to investigate the impact of Saps on the virulence potential of C. albicans during skin infections. The epidermal differentiation, skin permeability, and the protein and lipid metabolism of this tissue culture system have been shown to resemble the in vivo situation (Rosdy and Claus, 1990; Rosdy et al, 1993). Recently we

have established an in vitro model of cutaneous candidiasis for pharmacologic investigations (Korting et al, 1998; Schaller et al, 1999b). After infection with C. albicans, morphologic changes of the epidermis (scaling, hyperkeratosis, parakeratosis, dyskeratosis, spongiosis) corresponded to those found in established animal models and in vivo in man (Sohnle and Kirkpatrick, 1978; Odds, 1988; Sohnle and Hahn, 1992; Luna and Tortoledo, 1993). Candida albicans strain SC5314 was clearly able to invade the skin and caused marked epidermal damage. During the stages of cutaneous infection a progressive expression of SAP genes in the order SAP1 and SAP2 > SAP8 > SAP6 > SAP3 was observed. Although the same genes were expressed in the in vitro model for oral candidiasis (Schaller et al, 1998, 1999c) the order and preference seemed to be different. The development of initial edema and detachment of the super®cial corneocyte layers was accompanied by the expression of SAP1 and SAP2. This implies that these two Sap isoenzymes might preferentially contribute to the keratinolytic activity of C. albicans. Additional SAP8 transcripts were detected corresponding to deeper invasion of the yeast cells into the stratum corneum. Yeast to hyphal transition occurred when C. albicans cells penetrated into the stratum granulosum or deeper and was accompanied by SAP6 expression. The association between germ tube formation and SAP6 expression has been demonstrated before by northern analysis in in vitro studies (Hube et al, 1994) and by RT-PCR in a model of oral candidiasis (Schaller et al, 1998, 1999c). Expression of SAP3 in the later stages of infection was not accompanied by further visible morphologic alterations of the epidermis. In vivo investigations of candidiasis in patients infected with human immunode®ciency virus con®rmed secretion of Saps during oral infection (Schaller et al, 1999a). Further immunoelectron microscopy studies using two speci®c polyclonal antibodies directed against Sap1±3 or Sap4±6 con®rmed the secretion of speci®c Saps during different stages of the infection process. Candida albicans cells showed an intensive Sap1±3 and only little Sap4±6 labeling within the cell wall. The ultrastructural localization of Sap immunoreactivity within the cell wall has also been demonstrated previously during experimental rat vaginitis (Stringaro et al, 1997) and experimental oral candidiasis in an in vitro model (Schaller et al, 1999c). These results suggest that the majority of Saps during oral or cutaneous candidiasis are Sap1±3 and not Sap4±6. In contrast, a high production of Sap4±6 by C. albicans cells could be demonstrated after phagocytosis by murine peritoneal macrophages (Borg-von Zepelin et al, 1998). These results imply a direct role of Sap1±3 for tissue damage during super®cial infections whereas Sap4±6 seem to be important for interactions with cells of the immune system (Hube, 1998). We are aware that a possible role of Saps during interactions with cells of the immunosystem, as observed for SAP4±6 (Borg-von Zepelin et al, 1998), cannot be monitored in our in vitro model. The strong attenuation of the virulence phenotype after treatment with the speci®c aspartic proteinase inhibitor pepstatin A con®rms the contribution of Sap activity to tissue damage in this in vitro model of cutaneous candidiasis. A clear but not complete reduction of the morphologic alterations has been observed, which may imply an incomplete inhibition of Sap activity by pepstatin A. Incomplete in vitro inhibition of Saps by pepstatin A has also been demonstrated previously (Ollert et al, 1993; Colina et al, 1996; Korting et al, 1999; Schaller et al, 1999c). It is likely, however, that other virulence factors also contribute to tissue damage during Candida infections. Infection experiments with Dsap1 and Dsap2 demonstrated a reduced virulence phenotype compared with the parental strain infection. This con®rms the important role of these two isoenzymes for the initial stage of epidermal infection. In summary, these results illustrate that Saps contribute to tissue lesions in the in vitro model for cutaneous candidiasis. In addition, our data of a speci®c pattern of SAP gene expression suggest that distinct Sap isoenzymes are of relevance for skin infections.

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The authors thank J. Laude (Ludwig-Maximilians-University, Munich, Germany) for excellent technical assistance, M. Roecken and W. Burgdorf (LudwigMaximilians-University, Munich, Germany) for critical reading of the manuscript, and M. Monod (Center HoÃpitalier Universitaire Vaudois, Lausanne, Switzerland) for providing the polyclonal antibodies. The work was supported by grants from the Theodor-Nasemann-Stipendium and the Manfred-Plempel-Stipendium to M.S. This article is a part of the doctoral thesis written by Carolin Schackert at the medical faculty of the Ludwig-Maximillians-University Munich (in preparation).

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