Expression of Antimicrobial Peptides in Cutaneous Infections after Skin Surgery

M.R. Kesting; M. Stoeckelhuber; F. Hölzle; T. Mücke; K. Neumann; K. Woermann; F. Jacobsen; L. Steinstraesser; K.-D. Wolff; D.J. Loeffelbein; N.H. Rohleder

Disclosures

The British Journal of Dermatology. 2010;163(1):121-127. 

In This Article

Materials and Methods

Patients and Specimens

We investigated samples from male and female patients at the Maxillofacial Unit of the Technische Universität in Munich. The mean age of the patients was 57 years (range 18–79). The methods were approved by the local ethics committee (no. 212108) and are in accordance with the Helsinki Declaration (1975, as revised 1983). All the patients gave written informed consent.

Samples were collected from inflamed wounds (n = 27) at skin flap donor sites after reconstructive surgery of squamous cell carcinomas of the head and neck with microvascular flaps (excision from infected wound margin: 16 cervical/facial, four fibular, seven radial; Fig. 1). A wound was regarded as inflamed and included in the study when clinical signs such as redness, swelling and pain were evident and when a dehiscence occurred. Healthy tissue controls (n = 16) were collected from excess tissue from cosmetic surgery or residual flap donor site tissue (four cervical/facial, seven fibular, five radial; Fig. 2). From each patient, one half of the sample was placed in RNAlater (Qiagen, Hilden, Germany) and stored at −80 °C for PCR experiments, and the other half of the sample was stored in 4% buffered formalin for immunohistochemistry.

Figure 1.

Sample acquisition from infected wound margins. The dashed line indicates incision.

Figure 2.

Acquisition of healthy control samples from excess tissue during operation. The dashed line indicates incision.

RNA Isolation and Reverse Transcription

Samples were homogenized in a rotor-stator system (Miccra; ART Prozess- und Labortechnik GmbH, Müllheim, Germany). Total RNA was isolated by using the RNeasy® Protect Mini kit (Qiagen) following the manufacturer's instructions. The amount of extracted RNA was determined by measuring the optical density (BioPhotometer; Eppendorf, Hamburg, Germany). For the reverse transcription of 1 μg isolated RNA per sample, the SuperScript™ First Strand Synthesis System (Invitrogen, Karlsruhe, Germany) and random primers were used.

Real-time Reverse Transcription–Polymerase Chain Reaction

RT-PCR was performed in a LightCycler® 1.0 detection system (Roche, Mannheim, Germany) with 2 μL cDNA sample, 2 μL LightCycler® FastStart DNA Master SYBR Green I reaction mix (Roche), 1 μL of each forward and reverse primer (0·5 μmol L−1), 1·6 μL MgCl2 (3 mmol L−1) and 12·4 μL RNase-free water, resulting in a total volume of 20 μL per sample. Primers were selected from the Universal ProbeLibrary (Roche; sequences are given in Table 1). The specificity of the primers was verified by electrophoretic separation of the PCR products. Amplification algorithms were as follows: 10 min at 95 °C, 40 cycles of 15 s at 94 °C, 10 s at 60 °C (S100A7, 58 °C) and 10 s at 72 °C. A melting curve analysis was recorded in order to test for the consistency of cDNA fragments. The amount of RNA was automatically calculated by comparison of measured threshold cycles with an external standard curve. Data were normalized by determination of the amount of 18S rRNA. A no-template control was included in each run. All amplifications were carried out in triplicate.

Immunohistochemistry

The formalin-stored samples were embedded in paraffin. Sections of 4 μm were deparaffinized. After microwave irradiation for antigen unmasking, the slides were blocked with 3% H2O2 for 10 min and with goat serum (Vector Laboratories, Burlingame, CA, U.S.A.) for 45 min. Staining was performed with polyclonal primary antibodies against hBD-1 and -2 (1 : 100; Santa Cruz Technology, Santa Cruz, CA, U.S.A.), hBD-3 (1 : 100; Novus Biologicals, Littleton, CO, U.S.A.) and psoriasin (1 : 50; Imgenex, San Diego, CA, U.S.A.) according to the avidin–biotin–horseradish peroxidase complex method, by using the Vectastain® ABC kit (Vector Laboratories) at room temperature for 1 h and overnight at 4 °C. The slides were incubated with a biotinylated secondary antibody (1 : 200; Vector Laboratories) for 45 min (hBD-1, -2, -3) or 80 min (psoriasin). Then, the slides were incubated with peroxidase-conjugated streptavidin (Vector Laboratories) for 45 min. Diaminobenzidine was used as a chromogen. The sections were counterstained with haematoxylin. Negative controls, in which the primary antibodies were omitted, were treated identically. For all samples, stains were produced in duplicate. The sections were viewed with a Nikon Eclipse 80i microscope, and images were captured by a digital camera (Nikon, Düsseldorf, Germany).

Staining was scored independently by two investigators. For each sample, a staining intensity value (0, negative; 1, weak; 2, moderate; 3, strong) was assigned to the following epidermal layers: stratum basale (SB), stratum spinosum (SS), stratum granulosum (SG) and stratum corneum (SC).

Statistical Analysis

SPSS 16.0 software (SPSS Inc., Chicago, IL, U.S.A.) was used to perform statistical analysis. Differences in mRNA expression between the groups were analysed by the Mann–Whitney U-test. All P-values are local, given as two-tailed and are subject to a significance level of 5%.

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