Allergens and irritants transcriptionally upregulate CD80 gene expression in human keratinocytes.

The human CD80 costimulatory molecule is an important signal between professional antigen-presenting cells and T helper cells. The immunobiology of CD80 expression by keratinocytes, especially during allergic and irritant contact dermatitis, however, is less well understood. CD80 cell surface expression and gene transcription by keratinocytes was increased when keratinocytes were exposed to certain allergens (chemicals that induce inflammation via hapten-specific T cells) and irritants (chemicals that are toxic to epidermal cells). Therefore, the human CD80 promoter was cloned and luciferase reporter constructs containing various promoter fragments were engineered. Promoter mapping of these CD80 constructs in transiently transfected keratinocytes showed that a construct containing the proximal 231 bp immediately upstream of the transcription start site of the CD80 promoter was most active in keratinocytes and was inducible to a level ranging from 2- to 10-fold higher in keratinocytes treated with certain allergens and irritants, compared with untreated keratinocytes. This pattern of promoter fragment activity in keratinocytes is identical to that found in professional antigen-presenting cells. This is the first demonstration that the CD80 promoter is active in keratinocytes and that this activity is further increased in keratinocytes treated with certain allergens and irritants. These data suggest that allergens and irritants may, in part, break peripheral tolerance by their direct effects on keratinocyte costimulatory molecule expression, thereby facilitating interactions with epidermotropic T helper cells via the CD80-CD28 or CTLA-4 pathways.

Localization of the Darier disease gene to a 2-cM portion of 12q23-24.1.

Positional cloning with microsatellite markers allowed further localization of the Darier disease gene to a 2-cM interval of chromosome 12, 12q23-24.1, between the polymorphic loci D12S234 and D12S129. A region this size is suitable for construction of a contig to identify the Darier disease gene. Use of a polymorphic intronic marker for nitric oxide synthetase 1 gene, which maps to the same chromosomal area as the Darier gene, allowed exclusion of that gene as the Darier disease gene.

Localization of the gene for Darier disease to a 5-cM interval on chromosome 12q.

Darier disease is an autosomal dominant abnormality of epidermal differentiation characterized clinically by the presence of hyperkeratotic papules on the skin and histologically by the loss of cell cohesion and by disorderly keratinization. Two groups recently found evidence that the gene whose mutations underlie this disease is located at chromosome 12q23-q24.1, a site on chromosome 12 that clearly is distal to the type II keratin gene cluster. We report here evidence for sublocalization to a 5-cM region of that site in an additional ten families of European and Middle Eastern ancestry with a combined lod score in excess of 20.

Platelet indices: intra-individual variation in pre- and post-menopausal females.

Blood platelet counts and mean platelet volumes were determined at weekly intervals for eight weeks in 13 pre-menopausal and 11 post-menopausal females. Samples were analysed exactly two hours after blood collection on a Coulter S plus VTM particle counter. Pre-menopausal platelet counts were slightly higher than post-menopausal counts at each week, but the count was not affected by the menstrual cycle, intra-individual variation showing mean coefficients of variation (CV) of 7.4% and 5.2% respectively. The mean platelet volume was similar in pre- and post-menopausal groups at each week and showed little variability over time (mean CV 2.7% and 3.3%, respectively). Platelet parameters show little variability over time in either group and with no cyclical effects of menstruation apparent.

Interrelationship between platelet count, red cell count, white cell count and weight in men.

In 83 healthy normotensive males aged 20-55 years the platelet count is positively correlated with the red cell count (r = 0.371; P = 0.0006), the white cell count (r = 0.358; P = 0.0009), and with weight (r = 0.252; P = 0.0269). The red cell count is also positively related with the white cell count (r = 0.242; P = 0.0278) and with weight (r = 0.326; P = 0.0039); while the white cell count is slightly correlated with weight (r = 0.210; P = 0.067). These findings provide further indirect evidential support for a haemopoetic growth factor acting on a single pluripotent stem cell.

Racial differences in platelet counts in New Zealand men.

Healthy Maori males were found to have higher platelet counts (mean 295 x 10(9)/L: SD 46) compared to healthy nonMaori males (mean 253 x 10(9)/L; SD 50). Maori males had higher weights (mean 88.4 kg; SD 12.5) than nonMaori males (mean 81.0 kg; SD 9.0); and the platelet count was significantly correlated with weight (r = 0.30; p = 0.003). After weight adjustment significant differences between Maori and nonMaori males for platelet counts remained. These findings have implications for laboratory reference ranges, and may be of epidemiological significance in cardiovascular disease.

Counting reticulocytes by flow cytometry: use of thiazole orange.

Thiazole orange is a new fluorescent dye which will bind to the residual RNA in the cytoplasm of reticulocytes and allow their enumeration by FACS analysis. We have evaluated the use of this dye in the routine haematology laboratory. There is an excellent correlation between manual and FACS reticulocyte counts (r = 0.98) but FACS counting showed significantly higher precision (CV = 3.1) than the manual method (CV = 11.9) for single observer, 20.8% for multiple observers). Clinical specimens showed stable reticulocyte counts for 6 h if stored at 4 degrees C allowing efficient batching of samples. There was a significant fall in reticulocyte counts stored for 24 h at both 4 degrees C and 21 degrees C. Evaluation of 78 male and 76 female blood donors by FACS analysis gave normal ranges (mean % +/- 2 SD) of 0.74 +/- 0.48 and 0.84 +/- 0.56 respectively (P less than 0.005). When corrected to absolute values there was no sex difference (36 +/- 24 x 10(9)/l). Thiazole orange is an effective stain for the automated counting of reticulocytes by FACS analysis.

Long-term intra-individual variation of platelet parameters.

Mean platelet volume, platelet count and platelet distribution width were determined in eight male subjects at monthly intervals over a 4-6 month period. Measurements were made on a Coulter S Plus VTM exactly 15 min and 2 h after blood collection. Long-term intra-individual variability for mean platelet volume showed a mean coefficient of variation (CV) of 1.60% at 15 min and 2.01% at 2 h after blood collection; for platelet count a mean CV of 3.18% at 15 min and 2.40% at 2 h; and for platelet distribution width a mean CV of 7.3% at 15 min and 6.5% at 2 h. Platelet parameters are stable over time in the normal male individual.