Novel functional multiparameter flow cytometric assay to characterize proliferation in skin.

Keratins are a group of cytoskeletal proteins that are found in human epidermis and other stratified squamous epithelia. Several different types of keratins have been described. Keratin 10 (K10) is a keratin that is expressed in well differentiated, suprabasal keratinocytes, and keratin 6 (K6) is a keratin which is associated with hyperproliferation. Psoriasis is a chronic inflammatory skin disease, and besides inflammation, disturbed differentiation and hyperproliferation are its hallmarks. In order to study the hyperproliferation associated keratinization in both well differentiated and poorly differentiated keratinocytes, and in order to assess the proliferative activity of all K10 and K6 subpopulations, simultaneous assessment of K6, K10, and DNA content is required. So far, a triple staining protocol had not been available. In the present study, we established a novel protocol for simultaneous measurement of K6, K10, and DNA content, which enables the characterization of the proliferative activity of several cellular subpopulations in epidermis. From 16 patients with psoriasis and from 15 healthy volunteers, punch biopsies were obtained. After preparation of single cell suspensions, cells were stained with the anti-keratin 10 IgG(1)-isotype monoclonal antibody RKSE60, with the anti-keratin 6 IgG(2a)-isotype monoclonal antibody LHK6B, and with the DNA fluorochrome TO-PRO-3 iodide. Isotype specific secondary antibodies conjugated with phycoerythrein (PE) and fluorescein isothiocyanate (FITC) were used as the second step in the staining procedure. Controls were measured omitting the primary antibodies, and gates were set in order to differentiate between the K10 and K6 subpopulations. Samples from both psoriatic patients and healthy volunteers were than measured. Owing to the IgG specificity of RKSE60 and LHK6B, no cross-reactivity was observed between these antibodies. The triple staining with RKSE60, LHK6B, and TO-PRO-3 iodide showed subpopulations of K10 expressing cells, K10/K6 co-expressing cells, and K6 only expressing cells. There was a significant difference in the proportion of K6 expression and K10/K6 co-expression between psoriatic and normal skin. Moreover, the proliferative activity of these subpopulations could be quantified by this protocol. We concluded that a triple staining protocol for the assessment of K6, K10, and DNA content, using the monoclonal antibodies LHK6B, RKSE60, and TO-PRO-3 iodide, supplies reliable and reproducible data for cellular studies on these keratins and for studying the proliferative activity of the subpopulations of these keratins in epidermis. Moreover, the present study showed that with respect to the proportion of K6, significant differences are present between psoriatic and healthy human skin.

Conditions for the transition to home health care.

A number of changes in health care thinking have been taking place during the past years; these changes are in the financial system, in professional thinking and in political views. As one of the consequences, general hospitals in the Netherlands are thinking of entering home health care for professional and financial reasons. Most probably this may be done now if the management dares to take some risks. However, many conditions still need to be adapted to that situation. Most importantly, it is absolutely necessary to achieve a functional co-operation between professional groups in home health care. Hospital pharmacists should do their home work to make clear what extra value they can bring into home health care. Undoubtedly there are possibilities for the hospital pharmacy in home health care as part of the whole hospital policy in that direction.

[Vitamin D deficiency in femoral neck fractures (author's transl)]. / Vitamin-D-Mangel bei Schenkelhalsfrakturen.

In a therapeutic trial 20 patients with femoral neck fractures (FNF) were treated alternatively without supplemental vitamin D, calcium and phosphate or with 15 000 I.U. vitamin D2, 500 mg Ca++ and 560 mg of phosphate daily. Within two weeks, 25-OH-D in serum increased in patients with supplementary therapy. It is concluded that in the German Federal Republic, vitamin D deficiency is frequently found in the elderly during the winter months and that vitamin D deficiency could be important for the development of FNF.

Serum 25-hydroxycalciferol in myocardial infarction.

Oral intake of calciferol (vitamin D) was higher in patients with myocardial infarction than in controls. Having established an assay for 25-hydroxycalciferol (25-OH-D), the principal circulating form of the vitamin, we measured this compound in control subjects and patients with myocardial infarction. In controls, 25-OH-D varied with the season: levels were high in summer and low in winter. Furthermore, levels were low in control subjects above 60 years of age. In patients with myocardial infarction, normal and low values for 25-OH-D were found. It is concluded that in this region patients with myocardial infarction do not consume greater amounts of vitamin D.

[25-OH-vitamin D in the serum of patients with femoral-neck fractures]. / 25-OH-Vitamin D im Serum bei Patienten mit Schenkelhalsfrakturen.

In 10 women with femoral neck fracture, 10 with fracture(s) evolved from accidents (mainly traffic accidents) and 10 without disease (controls) in January and July, the 25-OH-vitamin D levels in serum were measured by a competitive protein binding assay. Patients with femoral neck fractures exhibited significantly (p less than 0.05) lower 25-OH-vitamin D levels than the other groups. Additionally, in patients with femoral neck fractures calcium x phosphate product in serum was subnormal (below 25). In Germany, prophylactic dietary supplementation of vitamin D seems feasible.