Introduction 4 9 31 35 40 52 3 5 10 13 15 24 25 2 32 46 30 33 37 38 57 61 62 7 9 29 30 33 34 47 50 8 11 12 18 26 27 36 39 41 43 54 19 22 42 48 19 22 + 19 20 58 51 36 6 20 27 49 58 59 The primary purpose of this study was to determine the expression of CCR5 and its ligands in chronic plaque psoriasis in situ compared to non-lesional skin, through analysis by immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). In order to examine the possibility that CCR5 plays a functional role in the pathogenesis of psoriasis, we also analyzed clinical and immunohistochemical data obtained from lesional and non-lesional skin biopsies of psoriasis patients before and after treatment with a CCR5 inhibitor. Materials and methods Study design and patients Lesional and non-lesional skin biopsies were obtained from nine patients with moderate to severe chronic plaque psoriasis, defined by the psoriasis area severity index (PASI) ≥ 8. These skin biopsies were analyzed by manual quantification of immunohistochemical double-staining and quantitative RT-PCR. In order to get insight in the possibility of a functional role of CCR5 in the pathogenesis of psoriasis, 34 patients, including the previous 9 patients, participated in an 8 week, randomized, placebo-controlled, parallel group, multi-centre, double-blind clinical trial in which patients received either 50 mg twice daily of the CCR5 inhibitor SCH351125 (23 patients) or matched placebo (11 patients) orally for 28 days, followed by a follow-up period of 4 weeks. During the follow-up period patients were only allowed to use emollients as treatment and on day 56 vital signs, PASI and blood were assessed. Patients were included at the dermatology outpatient departments of four academic hospitals from April 2004 to December 2004. At baseline and the last day of treatment (day 28), lesional biopsies were taken to evaluate the immunohistochemical effect of the CCR5 inhibitor. For this immunohistochemical evaluation, single-stained sections were analyzed with digital image analysis, semi-quantitative analysis (SQA) and confocal scanning microscopy, and double-stained sections on baseline and day 28 were analyzed by manual quantification. To evaluate the clinical effect of the CCR5 inhibitor the PASI was assessed at baseline, day 28 and day 56. ,  http://www.controlled-trials.com/ISCRT14986467 Biopsies Four-millimeter biopsies were taken from the inside border of a target psoriatic plaque, preferentially from a non-sun-exposed area. Lesional biopsies from each patient were obtained from the same target lesion, separated by at least 1 cm. The biopsy samples were randomly coded, snap-frozen in Tissue-Tek OCT compound (Sakura Finetek Europe, Zoeterwoude, The Netherlands) by immersion in liquid nitrogen and stored at −80°C until processing. Five-micrometer cryostat sections were cut and mounted on glass slides (Star Frost adhesive slides, Knittelgläser, Braunschweig, Germany), before being stored at −80°C until immunohistochemical staining. For each staining three sections of each biopsy were analyzed to minimize random variation. Immunohistochemistry + + + 2 RNA analysis RNA was extracted from frozen skin biopsies using the RNeasy mini kit (Qiagen). RNA quantity was assessed by OD at 260 nm and RNA quality was analyzed by measuring the ratio of 28s and 18s rRNA using the Agilent 2100 bioanalyzer. Quantitative PCR Taqman primers and probes were designed with Primer Express software (ABI), and purchased from ABI. The sequences of the human primers and probes are available upon request. For the human skin tissue, quantitative PCR was carried out with an ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The PCR reactions were prepared using the components from the Invitrogen Platinum Quantitative RT-PCR One-Step kit and assembled according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). The final concentrations of the primers and probe in the PCR reactions were 200 and 100 nM, respectively. The RT-PCR reactions for each gene were performed in a single 384-well plate. Separate plates of the same RNAs were used to quantitate 18S RNA as an internal control for RNA quality, and a primer/probe set for the CD4 promotor was used to check the RNAs for genomic contamination. The PCR data was quantitated based on a standard curve generated using fourfold serial dilutions of the target genes. The fourfold dilutions began at 0.25 ng, and eight dilutions were used to generate the standard curve. This procedure provides an absolute quantitation of the amount of CCR5 mRNA in a given tissue. Data were analyzed using Sequence Detection Systems software version 1.7 (Applied Biosystems, Foster City, CA, USA). Digital image analysis 23 2 Semi-quantitative analysis For keratinocyte expression of K16 keratin, a semi-quantitative score was done by two independent observers, blinded for order, patient and clinical data, with a standard binocular light microscope (Olympus) at 200× magnification. The semi-quantitative score ranged from 0 to 4+. A score of 0 represented no expression, while a score of 4 represented abundant expression in all layers of the epidermis. Confocal scanning microscope A E Sample size calculation The randomized placebo controlled clinical trial was targeted to randomize a total of 30 subjects (20 on active treatment and 10 on placebo). With this sample size, the trials would be able to detect a difference of 38% in the response rate from the placebo group assuming a 0% response rate in the placebo group with 80% power at an alpha level of 0.05 (two-sided test). Randomization Randomization was stratified by sites. Each site was assigned a fixed number of subjects numbers; e.g. site 1 would get numbers 1–9 and so on. Once the physician of the study site would enroll a subject, the subject would be assigned the next available subject number assigned to the site, starting with the bottom of the list; e.g. the first subject enrolled in site 1 would get number 1, the second subject would get number 2, and so on. Treatments would be assigned in an active to placebo ratio of 2:1 according to a computer generated randomization schedule. No stratification based on age, sex or other characteristics was performed. Throughout the study both patient and treating physician were blinded to the group assignment. Statistical analysis P t P Results Comparison of CCR5 expression in lesional versus non-lesional psoriatic skin 1 + + + + 1 + P + P Fig. 1 ns P P P  2 P P Fig. 2 P P  Lack of clinical efficacy of SCH351125: a CCR5 inhibitor 3 1 n 4 n Fig. 3 SCH351125  SAE AE  Table 1 Demographical data patients  Randomized clinical trial Placebo SCH351125 Number 11 23 Male:female 7:4 18:5 a 41.8 (10.2) 49.4 (14.3) a 20.6 (9.8) 19.8 (11.6) a 14.9 (4.7) 15.7 (4.3) a SCH351125 PASI BSA Fig. 4 SCH351125 PASI a b c d ns 1 P  In the treatment group four patients discontinued. One patient developed an erythrodermic eruption after 4 days of treatment, which was considered by the site physician as a serious adverse event (SAE). Two patients discontinued due to adverse events (AEs): one developed shingles in the n.trigeminus area of the right side of his face after 8 days of treatment and one patient discontinued due to hair loss after 21 days of treatment. One patient discontinued due to non-compliance. In the placebo group two patients discontinued due to AEs: both exacerbation of their psoriasis after 2 weeks of treatment. CCR5 expression before and after treatment with SCH351125 4 4 + + Discussion + + + + + + + 19 48 1 17 21 45 53 42 In summary, our results do not provide a clear answer to our objective of determining whether the percentage of CCR5 expressing cells is similar in lesional and non-lesional skin, or if this percentage is increased in lesional skin. 14 44 55 56 60 + + 4 16 21 27 28 49 26