Development and evaluation of a new PCR assay for detection of Pseudomonas aeruginosa D genotype.

This report describes a new PCR-based assay for the detection of Pseudomonas aeruginosa genotype D in occupational saturation diving systems in the North Sea. This genotype has persisted in these systems for 11 years (1993-2003) and represents 18% of isolates from infections analysed during this period. The new PCR assay was based on sequences obtained after randomly amplified polymorphic DNA (RAPD)-PCR analysis of a group of isolates related to diving that had been identified previously by pulsed-field gel electrophoresis (PFGE). The primer set for the D genotype targets a gene that codes for a hypothetical class 4 protein in the P. aeruginosa PAO1 genome. A primer set able to detect P. aeruginosa at the species level was also designed, based on the 23S-5S rDNA spacer region. The two assays produced 382-bp and 192-bp amplicons, respectively. The PCR assay was evaluated by analysing 100 P. aeruginosa isolates related to diving, representing 28 PFGE genotypes, and 38 clinical and community P. aeruginosa isolates and strains from other species. The assay identified all of the genotype D isolates tested. Two additional diving-relevant genotypes (TP2 and TP27) were also identified, as well as three isolates of non-diving origin. It was concluded that the new PCR assay is a useful tool for early detection and prevention of infections with the D genotype.

Expression of the psoriasis-associated antigen, Pso p27, is inhibited by cyclosporin A.

The psoriasis-associated antigen, pso p27, can be isolated from psoriasis scale and is present in complement-activating immune complexes in psoriatic scale, and in serum from patients with psoriasis. The antigen is produced by tryptase-positive cells in the skin lesions and is shown to be a major antigen in the immune reactions in psoriasis. The synthesis of this particular antigen is reduced with the remission of inflammation in the skin lesions. In this study we followed 3 patients with severe plaque psoriasis during treatment with cyclosporin A. In all patients we observed a decrease in the expression of the antigen pso p27 during the therapy. The effectiveness of the therapy varied among the patients, but there was a clear correlation between disease activity and expression of the antigen pso p27 as demonstrated by immunofluorescence in biopsies from selected skin lesions. This observation strengthens our hypothesis that the pso p27 antigen plays an important role in the inflammation in psoriasis.

Effects of cascade apheresis in patients with psoriasis and psoriatic arthropathy.

Eight patients with psoriasis, all with skin scales and 7 with disabling psoriatic arthritis, were subjected to cascade apheresis starting with three treatments per week for 2 weeks, followed by one treatment a week, comprising ten treatments in all. Six out of 7 patients (86%) with arthropathy and 3 out of 8 patients (38%) with scales experienced a beneficial effect. There was a large drop in the levels of circulating immune complexes (CIC) due to the treatment, and the removal of CIC was followed by reduced inflammatory activity in skin lesions and joints as evaluated by pain, morning stiffness, grip strength, plaque score, and PASI index. However, there was no correlation between the level of CIC, disease activity, or treatment response. From the present results it is concluded that CIC may play a more significant role regarding psoriatic arthropathy than in skin manifestations, and apheresis may be beneficial in patients not responding to conventional therapy.

Detection of psoriasis-associated antigen pso p27 in sarcoidosis bronchoalveolar lavage fluid using monoclonal antibodies.

Sarcoidosis is a multisystem granulomatous disorder of unknown etiology, predominantly affecting the lung. The increased concentration of pulmonary lymphocytes with specific receptors in subgroups of sarcoidosis patients suggests a local specific immune response. pso p27, a psoriatic scale antigen linked to the pathogenesis of psoriasis, was previously found in BAL cells, serum, and Kveim-Siltzback test in sarcoidosis. With an enzyme-linked immunoassay based on murine monoclonal antibodies, we analyzed BAL fluid from 21 patients with pulmonary sarcoidosis. Eleven (52%) of the patients have detectable levels of pso p27 antigen. No antigen is detected in the BAL fluid from five healthy, nonsmoking controls. Serum concentrations of pso p27 shows no significant difference between the two groups, but three of the sarcoidosis patients have detectable levels of the antigen. Mean concentration of pso p27 is >100 fold higher in BAL fluid than in serum from the sarcoidosis patients. This strongly suggests local pulmonary production of pso p27 antigen.

Use of scale antibodies for the detection of antigens in psoriatic lesions.

Extractable IgG from psoriatic scale was purified, labelled with biotin and used in ELISA and immunofluorescence (IF) in an attempt to detect and localize prominent antigens in psoriatic scale extracts and in psoriatic lesions, respectively. Biotinylated immunoglobulins isolated from psoriatic scale from each of 5 patients were used. Scale extracts were fractionated on a Sephacryl S-300 column, and antigens detected by scale IgG were eluted in the void volume and at a Kav 0.55. The profile was very similar for each antibody preparation. Antigens recognized by serum IgGs from both healthy controls and psoriatic patients were detected in the void volume only. Antigens recognized by a rabbit antiserum against the psoriasis-associated antigen, pso p27 (6), were restricted to the fractions eluted at a Kav 0.55. Furthermore, the binding of scale IgG to the antigens eluted at Kav 0.55 was inhibited by purified pso p27 antigen. Two of the scale antibody preparations gave rise to a distinct fluorescence on skin biopsies from psoriatic lesions in indirect immunofluorescence. The antigens recognized were localized to a subfraction of dermal cells and in the endothelial lining of some of the dermal vessels. Double labelling with these scale antibodies and a rabbit anti-pso p27 antiserum showed that both antibody preparations bound to the same cells in the psoriatic lesions, while only a minority of these cells were recognized by a murine monoclonal antibody against human IgG. The observations described indicate that the pso p27 is a major antigen in the immune reactions in psoriasis.

Surprisingly high levels of anaphylatoxin C5a des Arg are extractable from psoriatic scales.

Water-soluble extracts from psoriatic scales and normal human skin were prepared using either phosphate-buffered saline, pH 7.2, or 0.1 M carbonate buffer, pH 10.8. Anaphylatoxin C5a des Arg was quantified using a novel sandwich enzyme-linked immunosorbent assay (ELISA) using neoepitope-specific monoclonal antibodies. Alkali was about five to eight times more efficient than PBS in extracting C5a des Arg from scales, probably via dissociation of bound C5a des Arg. C5a des Arg concentration in scales from three patients suffering from psoriasis vulgaris varied between 2.5 and 4.6 ng/mg scale. No C5a des Arg was detectable in normal skin extracts. The biological activity of alkali-extractable C5a des Arg, i.e. chemotaxis, was preserved. The concentration of C5a des Arg relative to the concentration of albumin was taken as a parameter of the degree of complement activation in the psoriatic lesions, and was found to be more than six times higher than values attained in serum after maximum complement activation by zymosan. We conclude that complement activation may play a quantitatively important role in the inflammatory process in psoriasis.

An antigenic determinant is shared by psoriasis-associated p27 antigen and the Fc part of human IgG.

Rabbit antisera against the major internal protein, p27, of retrovirus-like particles from psoriatic urine, and against the serologically cross-reacting antigen, pso p27, from psoriatic scale, reacted with the Fc part of human IgG. Evidence indicating that the p27 antigen and the pso p27 antigen are identical has been presented in previous reports. A commercial antiserum against human IgG recognized a component in the pso p27-containing solution used as the source of antigen for immunization of the rabbits. By means of monoclonal antibodies against the pso p27 antigen, it was demonstrated that the Fc-reacting antibodies, and the antiserum against human IgG, recognized an epitope on the pso p27 antigen. The data indicated that an antigenic determinant is shared by the p27 antigen(s) and human IgG, suggesting that p27 antigen(s) may act as antigen(s) eliciting the production of antibodies with rheumatoid factor activity in psoriatic patients.