Allergic contact dermatitis and irritant contact dermatitis have different pathogenic mechanisms. It is therefore plausible that the epidermal expression of HLA-DR and the invariant chain associated with antigen processing and presentation might differ between allergic contact dermatitis and irritant contact dermatitis. We have quantified the volume of epidermal HLA-DR and invariant chain reactivity and the total epidermal volume in allergic contact dermatitis and irritant contact dermatitis using confocal laser scanning microscopy and indirect immunofluorescence on acetone-fixed 25 microns thick vertical skin sections. Eight nickel allergic patients were patch-tested with 5% nickel sulfate and 8 healthy volunteers were patch-tested with 4% sodium lauryl sulfate. Skin biopsy specimens were taken at 0, 6, 24, and 72 h after application of the patch tests. Sodium lauryl sulfate induced a statistically significant increased epidermal volume at 24 h and 72 h compared to 0 h and 6 h (p < 0.003 and p < 0.001, respectively), whereas an increase in epidermal volume in the allergic contact dermatitis group was not noted until 72 h after patch testing with nickel sulfate compared to 0, 6 h (p < 0.001) and 24 h (p < 0.004). No significant changes in the epidermal volume of HLA-DR reactivity were found at any time point within or between the two groups, nor was there any significant change in the epidermal volume of invariant chain reactivity in the allergic contact dermatitis group. In the irritant contact dermatitis group, however, the epidermal volume of invariant chain reactivity was significantly reduced from 17 +/- 8 x 10(3) microns 3 at 24 h to 9 +/- 3 x 10(3) microns 3 at 72 h (p < 0.04), which was also significantly lower than the 14 +/- 4 x 10(3) microns 3 observed in allergic contact dermatitis at 72 h (p < 0.01). Furthermore, the invariant chain expression was significantly lower than the HLA-DR reactivity in the irritant contact dermatitis group at 72 h (p < 0.001). The decrease of invariant chain reactivity at 72 h in irritant contact dermatitis might reflect an epitope-induced alteration by sodium lauryl sulfate or a down-regulated biosynthesis of the invariant chain due to variance in local cytokine production between allergic contact dermatitis and irritant contact dermatitis.

Background and objectives: Differences in blood sampling and separation techniques can affect the quantitative levels of activation markers on different leukocyte subsets. We examined the effect of two sampling procedures of EDTA blood on the quantitative levels of two markers, the CD11b/CD18 antigen and the EG2 epitope on intracellular eosinophilic cationic protein (ECP), in neutrophils and eosinophils, respectively. Materials and methods: Sample I was collected directly after completion of blood donation by an open technique and constant flow from the transfer tube directly into EDTA tubes. After sampling, the transfer tube was manually closed with a clamp. Sample II was collected 45 s later by the same technique by opening the clamp. Results: We found a significantly (p<0.01) higher expression of CD11b/CD18 on neutrophils collected by sampling procedure II than on those collected by sampling procedure I. In contrast, we did not find any difference in the intracellular ECP expression between sampling procedures I and II. To further explore the mechanisms for the observed upregulation of CD11b/CD18, fragments of a transfer tube were incubated with normal human serum (NHS) and heat-inactivated NHS (NHS56), respectively, for 60 min at +37 degrees C. Leukocytes from healthy blood donors were then incubated for 15 min at +37 degrees C with these serum preparations. The CD11b/CD18 expression was significantly higher (p<0.01) on neutrophils incubated with transfer-tube-activated NHS compared with NHS alone. However, when leukocytes were incubated with transfer tube activated NHS56, no difference was observed compared with incubation with NHS alone. In addition, by using confocal laser scanning microscopy, we could identify complement (C3c) deposits on the inner surface of the transfer tube fragments incubated in NHS, but not in NHS56. Conclusions: The quantitative level of the activation marker CD11b/CD18 on neutrophils, but not the EG2 epitope on intracellular ECP in eosinophils is significantly increased by a slight modification of the blood sampling procedure. It is suggested that the observed upregulation of CD11b/CD18 is caused by complement activation within the transfer tube. The results emphasize the importance of in-house data on the effect of variations in sampling procedures, particularly when data from healthy blood donors are included in clinical studies.

Background We have previously identified two major allergens of Pityrosporum orbiculare and characterized these as 37 kDa and 67 kDa proteins.Objective In the present study we have investigated the presence and subcellular location of the 37 kDa and 67 kDa allergen components in various members of the genus Pityrosporum as well as in Candida albicans, Candida parapsilosis and Saccharomyces cerevisiae.Methods To detect both cell surface and intracellular expression of the allergens, flow cytometry and confocal laser scanning microscopy (CLSM) were used. The cells were stained with indirect immunofluorescent (IIF) or alkaline phosphatase anti-alkaline phosphatase (APAAP) methods using mouse monoclonal antibodies (MoAbs).Results Ninety-five per cent of the P. orbiculare (P. ovale) cells cultured for 4 days showed cell surface-binding of the anti-37 kDa MoAb and 88% of the cells bound the anti-67 kDa MoAb when analysed with IIF and flow cytometry. It was found that the members of the genus Pityrosporum (Malassezia), P. pachydermatis and M. sympodialis, expressed the 37 kDa and 67 kDa allergens to a similar extent as did P. orbiculare. Less than 5% of the cells of the genus Candida and S. cerevisiae showed positive staining with the MoAbs. The CLSM revealed that the 37 kDa and the 67 kDa components were located to the cell wall and could not be detected inside the acetone fixed and APAAP stained yeast cells of the genus Pityrosporum. When the yeast cells were cultured for more than 4 days the expression of both allergens decreased significantly.Conclusion All three members of the genus Pityrosporum express the 37 kDa and 67 kDa major allergens on the cell surface, whereas these proteins could virtually not be detected in the Candida genus and S. cerevisiae.

Confocal laser scanning microscopy (CLSM) was used to localize the major allergen, Bet v I in birch pollen. Pollen grains of Betula pendula were collected from catkins that had begun to release their pollen, freeze dried, and stored at 4 degrees C. The pollen material was cryosectioned and stained with indirect immunofluorescence using the monoclonal antibody Bv 10. The observed localization of Bet v I was dependent on the treatment of the grains. When they had been prefixed with gaseous formaldehyde followed by acetone Bet v I was found in the cytoplasm. When the prefixation was omitted, a minor portion of Bet v I also appeared in the exine in the aperture regions. The allergen was not observed in the inline or the Zwischenkorper. Our suggestion is that the normal route for excretion of Bet v I is via the apertures on contact between pollen and the stigmatic surface of the pistil.

We used confocal laser scanning microscopy to analyze and compare Langerhans cells (LCs) in normal skin of six subjects. Acetone-fixed epidermal sheets and 25-microns vertical skin sections were incubated with fluorescein isothiocyanate-conjugated mouse monoclonal antibodies directed against HLA-DR. An individual threshold setting algorithm compensating for the differences in background fluorescence was applied to identify specific fluorescence. No statistically significant difference was found in the relative volume of epidermal HLA-DR reactivity between epidermal sheets (14 +/- 5%) and vertical skin sections (13 +/- 6%) or in the number of dendrites per HLA-DR+ LCs (7.8 +/- 3.1 and 5.9 +/- 3.1, n = 58, respectively). However, statistically significant higher background intensity was found in vertical sections than in epidermal sheets. Three-dimensional (3D) reconstructions of HLA-DR+ LCs revealed a concentration of HLA-DR to one or a few intracellular vesicles in 42 of 58 analyzed LCs in epidermal sheets and in 18 of 58 analyzed LCs in vertical sections. Direct contact between dendrites from different LCs was not found. The results indicate that both skin forms are suitable for quantitative studies. Owing to less background intensity and larger tissue volume, detailed 3D analysis of LCs is preferably performed on epidermal sheets rather than on vertical sections.

The dust mite Lepidoglyphus destructor is the dominating source of allergens giving rise to asthma and rhinitis among farmers. In a previous study of the localization of allergens in L. destructor we demonstrated that the 39 kDa allergen is associated with digestion. Here we describe the localization of the principal 15 kDa allergen and the high molecular weight allergen complex (79 and 93 kDa) in L. destructor with confocal laser scanning microscopy (CLSM). Cryostat-cut sections of mite bodies and faecal pellets were probed with mouse monoclonal antibodies (MoAbs) raised against the allergens. The 15 kDa allergen disclosed labelling of the mite body and most of the faecal pellets but left the exoskeleton unlabelled. The binding was widespread, and most intense in the mouth region. However, some staining was also observed around the gastrointestinal tract. In contrast, the 79 and 93 kDa allergen complex stained the exoskeleton and the front part of the mite. Interestingly, we detected no labelling of the faecal pellets with the MoAb against the 79/93 kDa allergen. The study indicates that the 15 kDa allergen is associated with the digestive tract whereas the function of the 79 and 93 kDa allergen complex remains to be elucidated.

We have analysed Langerhans cells (LCs) in basal cell carcinoma (BCC) and in healthy skin in 15 patients, using three different techniques: light microscopic examination of horizontal sheets, and of 6-mu m-thick vertical skin sections, and confocal laser scanning microscopy (CLSM) of 25-mu m-thick vertical sections. The use of CLSM enables both a quantitative and a three-dimensional (3-D) analysis of the cells in the same tissue volume. A statistically significant reduction in the relative volume of epidermal CD1a reactivity confined to tumour areas was found with CLSM. This difference was confirmed when the number of LCs in horizontal sheets were counted. In contrast, no significant reduction in epidermal CD1a(+) cells was found in thin vertical sections. This is probably due to the smaller tissue sample examined, and to variations in the number of CD1a(+) cells, with less cells directly overlying the tumour nests. The ratio of CD1a-expressing cells in the epidermis/dermis was significantly reduced in BCCs, compared with healthy looking skin. Few LCs were observed in tumour nests, but they were numerous in the surrounding stroma of the dermis. Three-dimensional reconstructions of CD1a(+) cells in BCC revealed striking morphological changes; they had a reduced number of dendrites, and these were often short and had few branches. The results demonstrate that CLSM is a suitable technique for quantitative and morphological analysis of CD la-expressing cells in the skin. We suggest that the alterations in LC numbers, distribution and morphology in BCC most probably are secondary to changes in the local environment.

Irritant contact dermatitis is a major problem in dermatology. One important group of substances causing irritant dermatitis is detergents. Exposure of the skin to detergents is frequent in both work and domestic environments. In the present paper we have studied how the penetration through the skin, and thus the effect, of the detergent sodium lauryl sulfate (SLS) is altered when the temperature is raised from 22 degrees C to 40 degrees C or 60 degrees C. We found that the penetration of sodium lauryl sulfate increased with increasing temperature. When comparing the increased penetration of sodium lauryl sulfate with the change in NiCl penetration at the same temperatures, we found that the increase in penetration was more pronounced for the detergent. This implies that the detergent also had a different effect on the structure and function of the epidermal barrier itself. The results underline the importance of choosing the right (low) temperature when working with detergent solutions to reduce the risk of developing irritant contact reactions.

Quantitative and detailed 3-dimensional (3-D) morphological information can be obtained from the same tissue volume using a confocal laser scanning microscope (CLSM). In the present study, we used CLSM for evaluation of Langerhans' cells (LC) in human skin at 0 h, 24 h and 48 h following occlusion with patch tests for 48 h. The relative volume of epidermal CD1a reactivity was quantified with CLSM on 25 microns thick sections stained with indirect immunofluorescence. No statistically significant difference was found when comparing the values obtained on the biopsy specimens from occluded skin (n = 36) with those from non-occluded skin (n = 9). Nor were any statistically significant changes detected in the number of epidermal CD1a+ cells as determined with immunoperoxidase staining between occluded and non-occluded skin. The occlusion produced a transient mild inflammatory reaction with an induced expression of intercellular adhesion molecule-1 (ICAM-1) on keratinocytes and an increased number of CD3+ epidermal lymphocytes. In addition, 3-D reconstructions revealed spatial information on the distribution of LC dendrites towards the skin surface.

Electron microscopes are not yet routine instruments in modern dermatological pathology even though they have provided detailed data about pathological changes in the skin for more than three decades. At present, dermatopathology is still dominated by light microscopy and especially so since the introduction of immunological techniques such as the use of monoclonal antibodies. These tools applied at electron microscopic resolution, however, may provide the ultimate identification of cells and subcellular components. In addition, electron microscopes have no peers in areas of quantitative investigation at subcellular levels, e.g. morphometry. The electron microprobe provides a unique tool in elemental analysis and may be used for the analysis of conventionally prepared specimens when foreign matter, not soluble in water, is deposited in the tissue. On the other hand, with water soluble substances the technique is most effective when freeze sections are utilized. This paper gives a selected review of the present day status of quantitative skin research as analysed with electron microscopy and the related technique of electron microprobe analysis.