Reduction of Langerhans cells in human epidermis during PUVA therapy: a morphometric study.

Skin biopsies were obtained before and after PUVA therapy from the normal skin of 5 psoriatic patients. By electron microscopic morphometric techniques the pretreatment volume density of Langerhans cells within the epidermis was estimated to be approximately 1%. This density was reduced in all 5 patients after therapy. The volume density of Langerhans cell granules per Langerhans cell cytoplasm was on an average 0.8% and this density was reduced in 4 of the patients after therapy, while in one patient there was an increase. There was a reduced volume density of Langerhans cell granules per unit volume of epidermis in all 5 patients after therapy. The mean length of the Langerhans cell disc was 260 +/- 120, nm the mean thickness 40 nm and the mean diameter of the Langerhans cell vesicle 120 +/- 30 nm. None of these parameters changed after PUVA therapy. The reduced volume density of Langerhans cells is discussed in the context of possible implications for immune reactions in the skin.

Scleredema adultorum associated with a monoclonal gammopathy and generalized hyperpigmentation.

Scleredema associated with a monoclonal gammopathy and generalized skin pigmentation is described in a 56-year-old man with hyperlipoproteinemia and cardiovascular disease. The patient had IgG-lambda paraproteinemia, without any evidence of multiple myeloma or immunoglobulin deposition in affected skin. Ultrastructural studies of pigmented lesional skin showed increased transfer of melanosomes to basal keratinocytes and dermal melanophages containing complex melanosomes. In addition, cytoplasmic, electron-opaque lipid droplets were seen in approximately every third keratinocyte or melanocyte, while only an occasional dermal cell contained lipid droplets. The hyperpigmentation appeared to be directly related to the scleredema, while the lipid deposition in skin was a likely consequence of the hyperlipoproteinemia. The findings further support the contention that paraproteinemia and hyperpigmentation may, in some patients, be associated features of scleredema adultorum.

Morphology and frequency of heterochromatic nuclear sheets in normal circulating human leucocytes and after PUVA therapy.

'Heterochromatic nuclear sheets' are structures that extend out from the nucleus into the cytoplasm where they are limited on both sides by the nuclear envelope. A uniform terminology regarding these structures has not been agreed upon and an attempt has been made in the present study to clarify the nomenclature. The frequency of nuclear sheets in circulating leucocytes has been established for the first time and found to be 4-8% for granulocytes and 1-2% for agranulocytes. Nuclear sheets have been reported to be associated with leukaemia, aneuploidy and treatment with antimitotic drugs. The parameter was therefore used to test the effect of photo-activated 8-methoxy psoralen (PUVA therapy) on circulating leucocytes. Used for the treatment of psoriasis PUVA therapy has been reported to cause leukaemia, chromosomal aberrations, mutations and skin cancer. No increase in heterochromatic nuclear sheets was found.

Increased size of stage II and III melanosomes during PUVA therapy.

Four different theories have been presented to show how early melanosomes are formed and at what stage in melanosome formation melanization starts. In the present investigation the perikaryon of melanocytes have been studied and the longitudinal and the transverse diameter of stage I-IV melanosomes estimated before and after PUVA therapy in 5 patients. An increase in the longitudinal diameter of stage II and III melanosomes was found, whereas no increase in stage I and IV melanosomes was detected. No increase in vesiculo-globular bodies was detected in stage I and II melanosomes after PUVA therapy, while in stage III melanosomes vesiculo-globular bodies appeared to have increased in number of PUVA therapy. It is concluded that during stimulated melanogenesis--as seen after PUVA therapy--an unknown mechanism is activated, causing increased amounts of structural proteins to be incorporated into each stage II melanosome. The vesiculo-globular bodies do not appear to take part in this process.

Intranuclear inclusions containing melanosomes observed after PUVA therapy: a study of five psoriatic patients.

Skin biopsies were obtained before and after PUVA therapy from five psoriatic patients and epidermal melanocytes surveyed for the presence of intranuclear melanosome-like bodies and nuclear bodies. Intranuclear melanosome-like bodies were observed after PUVA therapy whereas none were found before therapy. Nuclear bodies were found to increase in frequency after PUVA therapy. Remnants of a nuclear envelope membrane were not found around the intranuclear melanosome-like bodies. Nor were remnants of a nuclear envelope membrane found in or around the nuclear bodies. The results are discussed in view of the possible sites of origin of intranuclear melanosome-like bodies and nuclear bodies.

Erythrokeratodermia variabilis: immunohistochemical and ultrastructural studies of the epidermis.

Immunohistochemical and ultrastructural study of the epidermis was performed in a 53-year-old female with erythrokeratodermia variabilis (EKV) before and after treatment with the aromatic retinoid Etretinate (RO 10-9359). Aberrant expression of cytokeratin PKK2 (Labsystems, Helsinki, Finland) in lesional EKV stratum corneum was observed; this feature disappeared after Etretinate therapy. A normal distribution of DR-positive dendritic Langerhans' cells was seen in diseased, control and post-treatment skin specimens. The striking finding of this study was thus the shift to a basal cell-type keratin reactivity in stratum corneum in lesional skin, perhaps a reflection of cytoskeleton features related to cell adhesion. Increased adhesion between the cells in stratum corneum might account for the retention type of hyperkeratosis characteristic of EKV.

Ultrastructural studies on the effect of photoactivated 8-methoxy psoralen. Nuclear changes in a human epithelial cell line.

An established human epithelial cell line was exposed to photoactivated 8-methyoxy psoralen (PUVA); the cells were fixed and processed for electron microscopy 2 hours, 3 days and 6 days thereafter, and the nuclear morphology compared to controls. In the light microscope the cells fixed 3 and 6 days after PUVA exposure showed an increase in the number of multinucleated cells. At the ultrastructural level an increased number of cells with nuclear indentations, cytoplasmic invaginations, pseudoinclusions, nuclear bodies and multiple nucleoli were seen. An increased number of perichromatin granules per cell section and a decrease in heterochromatin situated marginally were observed. In cells from 6 day-old cultures 15 out of 50 nuclei were seen divided into 3-12 nuclear elements, compared with none in the controls. The smaller of these elements tended to be heterochromatic and the larger euchromatic. Generally the nuclear elements were smooth in outline, but elements of bizarre shape were also seen. These consisted of nuclear fragments united by heterochromatic extensions of uniform diameter. When comparing the morphology of PUVA exposed cells with the morphology of non-malignant and malignant cell lines, the PUVA exposed cells appeared to show an increase in morphological markers corresponding to those determined in malignant cells. The possibility that PUVA therapy may cause cancerous transformation is discussed.

Ultrastructural changes in the nucleus of a human epithelial cell line exposed to cytotoxic agents. The effect of PUVA and cadmium.

An established human epithelial cell line was exposed to cytotoxic doses of photo-activated 8-methoxy psoralen (PUVA) and to cadmium (Cd++) and the cells' nuclear ultrastructure were compared with controls. PUVA and Cd++ have some common biological features. Both substances induced nuclear indentations and cytoplasmic invaginations, while only PUVA caused an increase in nuclear bodies. After cadmium exposure, clusters of large-sized perichromatin granules (PG) were seen as well as an increase in chromatin margination. After PUVA, an increase in singularly distributed PGs was observed. The nuclear envelope was not affected by PUVA while Cd caused extensive disruption of this membrane. A strain of the epithelial cell line was made resistant to 100 mumol Cd/l and its nuclear ultrastructure was examined. An increase in chromatin margination was seen compared with its parent line. When this Cd resistant cell strain was exposed to 200 mumol Cd/l no ultrastructural changes took place. An attempt to produce a PUVA resistant epithelial cell strain was unsuccessful. Some functional aspects of the different morphological parameters observed are discussed.

The influence of different fixatives and a tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), on the induction of so-called dark cells in mouse epidermis. A light microscopical study.

Epidermal "dark cells" (DC) are believed to play a specific role in the so-called promotion phase of experimental skin carcinogenesis. They are recognized by their morphological features both at the light and the electron microscopical level. The possible effects of fixation on the morphology of epidermal cells and hence on the number of DC have not yet been thoroughly studied. In the present light microscopical study we used a semiquantitative method together with simple cell counting to evaluate the influence of fixation on the specific cellular morphology which is traditionally used to determine the number of DC. The use of cacodylate vehicled prefixatives, either formaldehyde or glutaraldehyde, led to a higher incidence of DC, and furthermore both to an increased width of the intercellular spaces (ICS) and a more heavy staining of the keratinocytes than when s-collidine vehicled glutaraldehyde was used. Differences in yield of DC solely due to the prefixative itself (formaldehyde or glutaraldehyde) were not detected. Exposure to TPA or the use of a hyperosmolal prefixative vehicle both yielded higher DC numbers than did controls or conventional prefixative vehicles, respectively. After prefixation with hyperosmolal vehicles, however, TPA treatment did not induce higher DC yield than in a control series. Phenomena usually accompanying exposure to TPA, such as intercellular oedema (widening of the ICS) and cytoplasmic vacuolization, varied in parallel to the number of DC. Hence, there is reason to believe that the induction of epidermal DC is mainly associated with volume reduction of keratinocytes. Such shrinkage may be due to the cytotoxic properties of TPA and degenerative phenomena appearing during tissue processing.

Characterization of a human epithelial cell line with special reference to its ultrastructure.

A commercially available cell line (NCTC 2544) originating from presumably normal human skin was chosen as an in vitro model system for subsequent studies of the effects of different agents on human epithelial cells. The cell line, therefore, was ultrastructurally and otherwise characterized at intervals by techniques which allow standardized controls of the model. The cell line was classified as epithelial both by phase contrast microscopy and transmission electron microscopy. The cells were polygonal, fully developed desmosomes were demonstrated and no extracellular filamentous material was observed. Specific epidermal markers like keratohyalin granules or keratinosomes were not demonstrated. Comparison of the nuclear morphology of cells from 1, 3 and 6 days old cultures revealed that only minor changes took place. The same was true for cytoplasmic features. Thus this cell line has a well-defined ultrastructural morphology. Flow cytometry studies showed the cell line to be tetraploid, with no sub-populations with other ploidies. Surface antigens were typed by a microcytotoxicity assay using 12 different antisera. The electrophoretic patterns of the isoenzymes lactate dehydrogenase and glutathione reductase were human. The cells did not form colonies in soft agar. Though several parameters indicate that this cell line is not a HeLa cell contaminant, this possibility cannot, however, be totally excluded.