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Many patients with moderate-to-severe plaque psoriasis do not respond adequately to methotrexate monotherapy. This pilot study, with a small patient population, was performed to evaluate the effectiveness and safety of etanercept and methotrexate combination in patients with plaque psoriasis and inadequate response to methotrexate. Outpatients with plaque psoriasis (Psoriasis Area and Severity Index > or = 8 and/or body surface area > 10%), despite methotrexate treatment (> or = 3 months; > or = 7.5 mg/week) were randomized to either etanercept with metho nottrexate tapered and discontinued (n = 28) or etanercept with continuous methotrexate (n = 31). Significantly more patients had a Physicians' Global Assessment of "clear"/"almost clear" in the combination group compared with etanercept/methotrexate taper (66.7 vs. 37.0%, respectively; p = 0.025). Adverse events were similar for both groups, with no cases of tuberculosis, malignancies or opportunistic infections reported. Addition of etanercept to methotrexate achieved significant improvement in psoriasis after 24 weeks.

The HCR gene, officially called Coiled-Coil alpha-Helical Rod protein 1 (CCHCR1), located within the major psoriasis susceptibility locus PSORS1, is a plausible candidate gene for the risk effect. Recently, CCHCR1 was shown to promote steroidogenesis by interacting with the steroidogenic acute regulator protein (StAR). Here, we examined the role of CCHCR1 in psoriasis and cutaneous steroid metabolism. We found that CCHCR1 and StAR are expressed in basal keratinocytes in overlapping areas of the human skin, and CCHCR1 stimulated pregnenolone production in steroidogenesis assay. Overexpression of either the CCHCR1*WWCC risk allele or the non-risk allele enhanced steroid synthesis in vitro. Furthermore, the cytochrome P450scc enzyme was expressed in human keratinocytes and was induced by forskolin, a known activator of steroidogenesis, and forskolin also upregulated CCHCR1. CCHCR1 has an altered expression pattern in lesional psoriatic skin compared to normal healthy skin, suggesting its dysregulation in psoriasis. We found that the expression of CCHCR1 is downregulated twofold at the mRNA level in cultured non-lesional psoriatic keratinocytes when compared to non-psoriatic healthy cells. Our results also suggest a connection between CCHCR1 and vitamin D metabolism in keratinocytes. The expression of the vitamin D receptor (VDR) gene was lower in non-lesional psoriatic keratinocytes than in healthy cells. Furthermore, Vdr expression was downregulated in the keratinocytes of mice overexpressing the CCHCR1*WWCC risk allele when compared to keratinocytes from mice with the non-risk allele of CCHCR1. Finally, we demonstrate that other agents relevant for psoriasis and/or the regulation of steroidogenesis influence CCHCR1 expression in keratinocytes, including insulin, EGF, cholesterol, estrogen, and cyclosporin A. Taken the role of steroid hormones, including vitamin D and estrogen, in cell proliferation, epidermal barrier homeostasis, differentiation, and immune response, our results suggest a role for CCHCR1 in the pathogenesis of psoriasis via the regulation of skin steroid metabolism.

The PSORS1 locus is the consistently replicated genetic risk factor for psoriasis. Clinical associations with the main marker allele of PSORS1, HLA-Cw6, have been addressed in a number of studies, but clinical associations have not been used as a way to distinguish the effects of the neighbouring candidate genes in PSORS1. Our results show that HLA-Cw6 and CCHCR1 risk allele associations with clinical features of psoriasis are predictably highly similar in a Finnish nationwide cohort of 379 psoriasis patients. The clinical profiling of a small group of patients (n=34) who were HLA-Cw6- but CCHCR1*WWCC positive suggested that no great differences existed between them and HCR-Cw6- patients. HCR+ genotype (as well as Cw6+ genotype) correlated for the first time positively with female sex and, in contrast with previous studies, negatively with disease severity. Presence of psoriatic arthritis was more pronounced in HCR- psoriasis (as well as in Cw6- psoriasis). Clinical profiling may be a useful approach to distinguishing genetic effects of candidate genes even within a locus in sufficiently large cohorts.

We have previously shown that HCR is a good candidate gene for psoriasis based on its location in the PSORS1 locus, predicted secondary structure change of the associated allele, and expression pattern. To understand better the function of HCR, we studied how HCR expression is altered in hyperproliferative skin diseases other than psoriasis and in cancers. We examined also its regulation by different cytokines, growth factors, and antipsoriatic agents using quantitative RT-PCR (TaqMan) analysis and its location by immunostaining of keratinocyte cultures. Compared to psoriasis, HCR protein had a different distribution in chronic dermatitis, pityriasis rubra pilaris, mycosis fungoides, and chronic skin ulcers. In three of six grade III squamous cell carcinomas of the skin, four of four adenocarcinomas of the lung, and two of two ductal breast adenocarcinomas, positive cytoplasmic staining in cancer cells was detected. As in psoriasis, Ki67 did not colocalize with HCR. In cell cultures, HCR staining was detected perinuclearly in the cytoplasm and in the nuclei, suggesting that the protein may have a role in both compartments. A 2-fold downregulation of HCR mRNA expression was observed on stimulation with interferon-gamma. Based on the observations that HCR is detected in cancers of epithelial origin in Ki67-negative areas and that interferon-gamma downregulates its expression, we suggest it to have an antiproliferative function.

The major susceptibility locus for psoriasis, PSORS1, resides on chromosome 6p and includes the candidate genes HLA-C, HCR, and CDSN. Based on a nationwide collection of psoriasis patients and genotyping for the PSORS1 susceptibility haplotype, we selected for a genome scan nine families who do not show association with PSORS1 to more easily detect minor loci for psoriasis susceptibility. In the genome scan, five loci gave initial evidence of linkage and were studied with a denser marker map. After fine mapping, only one locus on 18p11.23 showed suggestive evidence of linkage (nonparametric multipoint linkage analysis score, 3.58; p = 0.0038). The bootstrapping analysis showed that one large family contributed the majority of the linkage (p = 0.0039), but was supported by other families. Haplotype sharing between the linked families and haplotype association analysis gave additional support for the locus. Further, the 18p locus has shown nominal evidence of linkage with psoriasis in the British population. Taken together, these findings confirm the presence of a minor susceptibility locus for psoriasis on 18p11.

Keratinocyte hyperproliferation, inflammatory infiltrates, neoangiogenesis and alterations in cytokine levels are hallmarks of psoriatic skin. Matrix metalloproteinases (MMPs) have been associated with the remodeling of the extracellular matrix during inflammation, neovascularization, and malignant transformation. We have previously shown that particularly MMP-12 is abundantly expressed by macrophages and MMP-9 in macrophages and neutrophils of psoriatic lesions. In this work the expression of two novel metalloproteinases, MMP-19 and MMP-28, was investigated in psoriatic lesional and non-lesional skin. MMP-19 protein was detected by immunohistochemistry in 28/29 samples in keratinocytes in the same regions as Ki67 (marker of proliferating keratinocytes) and p63 (marker of keratinocyte stem cells). Immunosignaling was also seen in endothelial cells and fibroblasts. Furthermore, MMP-19 mRNA was upregulated in psoriatic keratinocytes and skin as assessed by quantitative real-time polymerase chain reaction. In lichen planus and lichenoid chronic dermatitis, MMP-19 staining was found in keratinocytes in areas where the basement membrane was abnormal. MMP-28 was not detected in psoriatic or non-lesional skin. Our results suggest that keratinocytes as well as the previously reported cell types (smooth muscle, endothelial and macrophages) can express MMP-19 in psoriasis and lichen planus. Upregulation of MMP-19 in keratinocytes may be influenced by changes in the architecture of the basement membrane zone.

Capacitative calcium entry and calcium wave propagation were studied in keratinocytes cultured from control persons and patients with type 1 neurofibromatosis. The cells were stimulated mechanically in the presence of inhibitors of gap-junctional or ATP-mediated communication to determine which pathways are operative in Ca(2+) signaling between these cells. Keratinocytes cultured from patients with type 1 neurofibromatosis (NF1) had a tendency to form cultures with markedly altered calcium-related signaling characteristics. Specifically, the resting Ca(2+) levels, intracellular Ca(2+) stores, capacitative calcium influx, and gap-junctional signal transduction were defective in NF1 keratinocytes. Western transfer analysis revealed apparently equal connexin 43 protein levels in normal control and in NF1 keratinocytes. Indirect immunofluorescence, however, demonstrated that connexin 43 was relatively evenly distributed in NF1 cells and did not form typical gap-junctional plaques between keratinocytes. Furthermore, the speed of the calcium wave was reduced in NF1 cells compared to normal keratinocytes. The results demonstrate that keratinocytes cultured from patients with NF1 display altered calcium-mediated signaling between cells.

Summary: We have previously shown that NF1 (type 1 neurofibromatosis) p21ras GTPase-activating tumor suppressor protein undergoes major relocalization during the formation of cell-cell junctions in differentiating keratinocytes in vitro. This prompted us to study the distribution of NF1 mRNA under the same conditions by in situ hybridization. In differentiating keratinocytes, the NF1 mRNA signal intensified within the cell cytoplasm within the first 0.5 to 2 hours after induction of cellular differentiation. First, the hybridization signal was evenly distributed throughout the cytoplasm. Subsequently, NF1 mRNA was gradually polarized to the cellular periphery at the side of cell-cell junctions and finally disappeared. Reappearance of NF1 mRNA was found in migrating keratinocytes forming a bilayered culture. Disruption of microfibrillar cytoskeleton, but not microtubules, caused a marked change in the subcellular distribution of NF1 mRNA. This data may suggest that intact actin microfilaments are essential for transport of NF1 mRNA to the cell periphery. This is the first study demonstrating that NF1, or any tumor suppressor mRNA, belongs to a rare group of mRNAs not targeted to free polysomes or ribosomes of the rough endoplasmic reticulum. This finding recognizes a potential way for post-transcriptional modification of NF1 expression.

This study demonstrates the presence of tight junction antigens in adult and developing human epidermis. Indirect immunofluorescence labeling and immunoelectron microscopy with antibodies to ZO-1 and occludin localized tight junction components ZO-1 and occludin to a narrow zone of the granular cells of adult epidermis. Double immunolabeling for tight junction components with adherens junction or desmosome proteins suggested that occludin is more specific for tight junctions than ZO-1, which may also be associated with adherens junctions. In developing skin, tight junctions interconnected the peridermal cells, and after the fetal stratification localized to the granular cell layer. Immunolabeling of psoriasis, lichen planus, and ichthyosis vulgaris, representing aberrant differentiation of the epidermis, showed that these conditions were associated with relocation of ZO-1 and occludin to the spinous cells. Cultures of epidermal keratinocytes, which offer a useful model for the formation of cellular contacts, revealed that tight junction components, ZO-1 and occludin, displayed a marked degree of colocalization relatively late during the process when the fusion zone had assumed a linear appearance. This suggests that the formation of adherens junctions and desmosomes precedes that of tight junctions. We speculate that the epidermal barrier, isolating the human body from the external environment, is in part formed by tight junctions of stratum granulosum.

Intracellular calcium plays an important part in the regulation of proliferation and differentiation of keratinocytes. Detached from their in vivo environment, cultured psoriatic keratinocytes were investigated by monitoring free intracellular calcium concentration, which was measured using fura-2/AM as a calcium-sensitive probe. The mean increase in intracellular calcium of psoriatic keratinocytes was significantly reduced compared with control keratinocytes when intracellular calcium stores were mobilized from endoplasmic reticulum with thapsigargin. This finding suggests defective capacitative calcium influx of psoriatic cells. Intracellular calcium stores were similar in psoriatic and control keratinocytes, when extracellular calcium was chelated with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid and intracellular calcium was depleted with thapsigargin. Mechanical wounding of keratinocyte monolayer resulted in a significantly reduced rise in intracellular calcium of psoriatic cells in low (< 0.1 mM) and high (1.8 mM) extracellular calcium suggesting defective intercellular coupling of psoriatic keratinocytes. Blocking of gap-junctions with heptanol in wounded keratinocytes did not affect the intracellular calcium response in psoriatic keratinocytes in contrast to healthy keratinocytes. Adding adenosine triphosphate to culture medium resulted in a more pronounced intracellular calcium increase than thapsigargin in psoriatic keratinocytes, suggesting that inositol triphosphate-mediated, P2-purinergic signaling was enhanced in these cells. Moreover, psoriatic keratinocytes maintained their defective responses up to at least fifth passage suggesting that psoriatic keratinocytes have an inborn error in calcium metabolism, rather than a localized defect in response to altered extracellular calcium gradient observed in vivo.

The expression and subcellular localization of neurofibromatosis type 1 tumor suppressor was studied in keratinocytes induced to differentiate by increased Ca2+ concentration of the culture medium. Differentiating keratinocytes became intensely immunoreactive for neurofibromatosis type 1 protein, which was apparently associated with cellular fibrils. Double immunolabeling with antibodies to cytokeratin 14 and neurofibromatosis type 1 protein suggested an association of intermediate type cytoskeleton and neurofibromatosis type 1 protein. The presence of neurofibromatosis type 1 protein in cell preparations treated with cytoskeletal buffer indicated a high affinity interaction between intermediate filaments and neurofibromatosis type 1 protein. Further studies utilizing double immunolabelings revealed that the intense neurofibromatosis type 1 tumor suppressor signal on intermediate filaments was temporally limited to the period in keratinocyte differentiation in which the formation of desmosomes takes place. Keratinocytes were also cultured from nine patients with type 1 neurofibromatosis and were studied with respect to cell morphology, and association of neurofibromatosis type 1 protein with intermediate cytoskeleton. The results showed that keratinocytes cultured from patients with neurofibromatosis type 1 displayed a highly variable cell size and morphology compared to controls. The latter findings represent predicted alterations in a situation where cytoskeletal organization is disturbed. Furthermore, differentiating neurofibromatosis type 1 keratinocytes were characterized by a reduced number of cytokeratin bundles that were decorated neurofibromatosis type 1 protein. The results of this study suggest that neurofibromatosis type 1 tumor suppressor exerts its effects in part by controlling organization of cytoskeleton during the formation of cellular contacts.

Acne fulminans is a rare, severe type of acne with unknown etiology. Ulcerative acne lesions, fever and musculoskeletal pain are typical symptoms. In addition, osteolytic or even destructive osteomyelitis-like bone lesions occur in many patients with acne fulminans. In the present study the degradation product (ICTP) of type I collagen, the most abundant collagen of the skeleton, was measured from the sera of patients suffering from acne fulminans. In 3 of 4 acne fulminans patients with active disease, the ICTP concentrations were clearly higher than the range of concentrations in age-matched controls. The mean concentration of ICTP in the acne fulminans patients was 17.6 +/- 6.0 micrograms/I, whereas the corresponding concentration in 6 patients with severe nodular acne was 6.9 +/- 2.1 micrograms/I. Increased uptake of radionuclide in bone scans was observed in all of the 4 patients with acne fulminans. The present results suggest that ICTP is increased in acne fulminans, due to the destruction of bone collagen matrix. ICTP could thus be used for monitoring the activity of acne fulminans affecting the skeleton.

Neurofibromin enhances the inactivation of protooncogene p21ras and has been suggested to function as a regulator of cell growth and differentiation. In normal skin, neurofibromin is particularly abundant in the basal keratinocytes of epidermis. The present study utilized antibodies raised against two synthetic peptides corresponding to different regions of neurofibromin. One of the antibodies recognized all forms of neurofibromin and the other was specific for type II neurofibromin. The following specimens were analyzed for neurofibromin immunoreactivity: 1) skin of apparently healthy volunteers, 2) active lesions of 15 psoriatic patients, 3) apparently healthy skin of the same patients at the time of the active phase of the disease, and 4) the previously lesional areas after anti-psoriatic treatment of the same patients. The presence of neurofibromin mRNA in normal epidermis and in keratinocytes cultured from normal skin was demonstrated by reverse transcriptase-polymerase chain reaction or by Northern hybridization. In marked contrast to normal epidermis, active psoriatic lesions were characterized by a weak immunosignal for types I and II neurofibromin in the basal cell layer of the epidermis. Previously lesional, clinically healed areas displayed variable, yet clearly detectable, expression of neurofibromin. Our results demonstrate that the epidermis of psoriatic lesions displays reduced immunostaining for type I and II neurofibromins compared to normal epidermis, and that neurofibromin immunoreactivity is partially restored concomitant with clinical healing of the lesions. The question whether the changes in neurofibromin expression in psoriasis are causal or consequential with respect to the pathogenesis of psoriasis remains to be elucidated.

To investigate the inflammatory and immunological aspects of severe acne, we examined the luminol-enhanced chemiluminescence of whole blood, T-cell subsets and natural killer cell functions in 11 patients with severe nodular acne and 4 patients with acne fulminans. In patients with severe nodular acne, the active phase of the disease, compared to the values in remission (means 47 mV, SD 24.8 and 32 mV, SD 8.3, p < 0.05). The patients with acne fulminans also showed high values in the active phase of the disease (mean mV 68.3, SD3.5) compared to remission (mean 30.5 mV, SD 15.3). No marked alterations were seen in the percentages of T-helper cells, T-suppressor cells or DR-positive lymphocytes or in the levels of soluble interleukin 2 receptor. The percentages and activities of natural killer cells did not show any significant changes either. Five patients (4 with severe nodular acne and one with acne fulminans, accounting for 33% of all patients) carried HLA Cw6 antigen, which is a significantly increased frequency compared to health controls (pc = 0.015). The present chemiluminescence results suggest that peripheral blood neutrophils are activated in patients with severe acne.

Prednisolone combined with erythromycin was given to 6 patients with cystic acne. The treatment responses were compared to those in 6 patients with cystic acne receiving isotretinoin and erythromycin and also to those in 3 patients with acne fulminans treated with prednisolone and erythromycin. During the first 4 weeks cystic acne showed a clear improvement in 5 out of 6 patients in both treatment groups. A similar improvement occurred in all 3 patients with acne fulminans. When corticosteroid was stopped, 2 out of 5 patients with cystic acne had a relapse and needed isotretinoin for complete control. In the isotretinoin-treated group, one patient with cystic acne needed prednisolone because the acne worsened to an ulcerative form. Slightly elevated liver enzymes, possibly due to erythromycin treatment, were observed in 2 patients with cystic acne and in one patient with acne fulminans. The present results show that prednisolone combined with erythromycin is an effective treatment during the early stages of cystic and febrile acne, but isotretinoin is needed for long-term control.

We examined all babies born live (4346) at two Finnish hospitals in the course of one year to determine the frequency of birthmarks, specially pigmented lesions, among Finnish newborns. All birthmarks excluding common salmon patches on the forehead and neck were recorded and photographed at birth. The babies were re-examined at the age of three months. Various birthmarks were recorded for 241 of 4346 babies, i.e. for 5.5% of all newborns. Ninety-one (2.1%) infants had congenital pigmented skin lesions, 167 (3.8%) had various vascular lesions and 21 (0.5%) had other birthmarks. The frequency of congenital melanocytic naevi was 1.5%. Most of the naevi were less than 20 mm in diameter. Only one child had a giant naevus. The frequency of congenital naevi in our study was the same or somewhat higher than previously described (1-8) but fewer other pigmented skin lesions were found than in previous studies perhaps due to racial differences.