Effects of 1-year intermittent treatment with topical tacrolimus monotherapy on skin collagen synthesis in patients with atopic dermatitis.

BACKGROUND: Topical corticosteroids decrease collagen synthesis during short-term treatment and can induce skin atrophy when applied over the long term. In contrast, short-term tacrolimus ointment therapy does not affect collagen synthesis. OBJECTIVES: Our aim was to evaluate the long-term effects of 0.1% tacrolimus ointment on collagen synthesis and on skin thickness in adults with moderate to severe atopic dermatitis (AD) and to compare the findings with the effects of conventional steroid-based therapy. METHODS: Fifty-six patients with AD were treated with 0.1% tacrolimus ointment in a 1-year, open-label, prospective clinical trial. Thirty-six patients with AD applied conventional steroid-based therapy and 27 healthy subjects were recruited as controls. The primary endpoint was the change in levels of procollagen propeptides I and III measured by radioimmunoassay between baseline and month 12. Additional endpoints included the change in skin thickness measured by ultrasound between baseline and month 12. RESULTS: Procollagen propeptide baseline values were significantly lower in the group to be treated with tacrolimus ointment than in healthy controls. One-year treatment with tacrolimus ointment was associated with an increase in collagen synthesis; the median increase in combined procollagen propeptide levels was 272 micro g L-1 (+ 140.9%, P < 0.001) and was accompanied by a significant increase in skin thickness. In three patients with visible skin atrophy, this condition ameliorated. Corticosteroid-based therapy had no significant effect on collagen synthesis; the median increase in combined procollagen propeptide levels was 11 micro g L-1 (+ 3.9%). A significant reduction in skin thickness was demonstrated. CONCLUSIONS: Long-term tacrolimus ointment therapy in patients with AD is nonatrophogenic and reverses corticosteroid-induced skin atrophy.

Tacrolimus ointment does not affect collagen synthesis: results of a single-center randomized trial.

We conducted a randomized, double-blind, placebo-controlled trial to assess the atrophogenicity of tacrolimus ointment. In a combined group of atopic dermatitis patients (n = 14) and healthy volunteers (n = 12), 0.3% tacrolimus, 0.1% tacrolimus, betamethasone-valerate, and a vehicle control were applied in a randomized order to nonsymptomatic, 4 cm x 4 cm regions of abdominal skin. After 7 d of treatment under occlusion, the carboxy- and amino-terminal propeptides of procollagen I (PICP, PINP) and the amino-terminal propeptide of procollagen III (PIIINP) were measured from suction blister fluid with specific radioimmunoassays. In addition, ultrasound measurements of skin thickness were taken. Betamethasone-treated areas showed median PICP, PINP, and PIIINP concentrations of 17.0%, 17.6%, and 39.5% of the vehicle control at the end of the treatment period, respectively, whereas the 0.1% and 0.3% tacrolimus-treated areas showed median concentrations of approximately 100% of the vehicle control (p < 0.001). Betamethasone was also the only treatment to reduce skin thickness; the median decrease in skin thickness was 7.4% relative to 0.1% tacrolimus, 7.1% relative to 0.3% tacrolimus, and 8.8% relative to the vehicle control (p < 0.01). Results for atopic dermatitis patients and healthy volunteers were similar. These findings suggest that tacrolimus does not cause skin atrophy.

A programme for assigning target values for external quality assessment schemes in countries with no authorized reference laboratories. Annex. Experiences with deviating results on Ektachem 700 XR.

The use of consensus values in external quality assessment schemes (EQAS) involves several problems and should preferably be replaced with target values obtained by methods of high metrological level. However, such values are difficult to obtain. In the present study we transferred values from the NIST (former NBS) certified reference serum SRM 909 to lyophilized and frozen test sera for various inorganic components using flame absorption or flame emission spectrometry. Enzyme values were assigned by laboratories of members of the former Scandinavian Enzyme Committee. The assignment was based on 2-4 determinations each day through 3 days of experiment. A total of 10 laboratories participated in the work. The results were utilized in a Danish EQAS. One practical concern is the fairly long time (9 months) which was needed for production, collection and compiling all data. To get an impression of how much dry chemistry analysers, e.g, could influence consensus values a Kodak Ektachem 700 XR was studied using lyophilized and frozen sera. The results are reported in the annex. On NIST SRM 909 the values found for sodium(I) were 6% too high even though the findings on frozen human sera were accurate. For aspartate aminotransferase a result three times the target values was found on a human lyophilized serum, while the values on the frozen sera only were slightly too high.

A Nordic reference serum suitable for use as trueness control in the clinical routine laboratory.

The described reference serum is characterized by: liquid human serum at "normal" level stored in frozen state at -80 degrees C; minimum damage of proteins; aseptic preparation; cryoprecipitate and excess fibrin removed; serum cleared by ultracentrifugation; pH at 7.2-7.6; available in sealed glass ampoules with inert gas (one ml serum in each); specified components among most frequently analyzed analytes; homogeneity assured and stability monitored; produced under strict rules for good manufacturing practices (GMP). The assigned values are traceable to reference measurement procedures and reference materials of highest achievable metrological level; according to the present proposal the maximum allowable uncertainty of the assigned value is based on biological variation (shared common reference intervals); the uncertainty should ideally not exceed 1/5 of the maximum allowable bias of results obtained on patients samples (even 1/2 would theoretically be acceptable and, for a practical guide approximately < 1% may suffice). The present document provides some guidance of how the reference serum could be established in practice. The document also indicates the use of the material and further extension of the concept. The present work is done as a NORDKEM project.

A possible model for accuracy control of determination of serum cholesterol with use of reference methods. A NORDKEM project.

A candidate definitive method for determination of total serum cholesterol developed at the Center for Analytical Chemistry, National Bureau of Standards, USA, has recently been compared with a reference method based on isotope dilution-mass spectrometry, developed at Huddinge Hospital. There was no significant difference (0.2%) in results obtained with the two methods. The Huddinge method was used here for assessment of inaccuracy of a defined enzymatic method, set up and used at four laboratories, one in each of four Nordic countries. The results obtained in the analysis of 21 patient samples were not significantly different from those obtained with the isotope dilution method. In the analytical range 1.7-14.2 mmol/1, the regression equation for the defined enzymatic method (y) versus the isotope dilution method (x) was y = 0.994x - 0.06 and the correlation coefficient 0.9999. The mean between-laboratory variation for the defined enzymatic method was 2.3%. The possibility is discussed that the defined enzymatic method can be used as candidate secondary reference method for assessment of inaccuracy of field methods in national quality control activities. In such a control system, the accuracy can be traced back to the definitive methodology through the hierarchical system.

Purification and heterogeneity of human kininogen. Use of DEAE-chromatography, molecular sieving and antibody specific immunosorbents.

Various methods of preparing human kininogen were investigated with an aim to limit the immunoreactive contaminant proteins to permit purification by immunosorption. A five-step procedure is described giving 7.5% yield of highly purified kininogen (pharmacological purity 14--20) from pooled human plasma, and containing approximately 30% alpha-2HS-glycoprotein and 2.8% albumin. Alpha-2HS could not be removed by polyacrylamide gel electrophoresis or isoelectric focusing in column. Analysis of heterogeneity of kininogen after chromatography on DEAE-Sephadex using various linear gradients and gel filtration on Sephadex G-100 suggested that a minor component may be an aggregate, not included in the yield. It remains uncertain whether this component derives from an occasionally observed high molecular form of active kininogen in the primary purification steps in the 7-12 S sieve fractions from Sephadex G-200, and excluded from further purification by pooling. Purification with immunosorbents was investigated using batch operations with antibody specific polymers prepared with antisera insolubilized with ethylchloroformate. It was found that the adsorption-desorption procedure was favourable for immunization purposes in producing highly specific immunologically pure kininogen. The kininogen obtained by this method or by the removal of contaminant alpha-2HS and albumin with the corresponding antibody specific polymers gave similar heterogenous patterns by polyacrylamide gel electrophoresis, indicating a main band of kininogen and several faintly stained bands which responded only to anti-kininogen. With 200 mug of the kininogen protein purified by immunosorption using monospecific antiserum the kininogen precipitation titre was 1:8 after 6--8 weeks in rabbits. With a polymer prepared with 4 ml anti-kininogen serum (1:8) and incubated with 800 mug highly purified kininogen approximately half the protein was desorbed with 2 M and 3 M sodium iodide in the first adsorption-desorption procedure.