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The acid phosphatase activity (acP) of developing skin window macrophages topically treated with 6-methyl-prednisolone (6-MP) has been evaluated by a quantitative-cytochemical technique (SMP 05/PDP 12-20). The following conclusions can be drawn: 1. The degranulation of migrated monocytes can be inhibited by local application of 0.1 to 10.0 micrograms of 6-MP per skin window lesion. 2. The increase of acP-activity associated with the physiologic transformation of monocytes into macrophages and the concomitant activation of the lysosomal system was clearly inhibited by 0.05 to 10.0 micrograms per skin window lesion. 3. 6-MP alters the morphology of the developing macrophages and alters the intracellular distribution of acP. 4. It can be concluded that 6-MP inhibits the development of monocytogenic macrophages. This effect is observed with amounts of 6-MP which correspond to those obtained by systemic application of 6-MP in usual dosages. The antiphlogistic effect of 6-MP probably is due to a considerable proportion by the direct influence on the inflammatory cells.

A modified technique for demonstration of acid alpha-naphthylacetate esterase was examined with regard to specificity and application on human T-lymphocytes in peripheral blood and pleural fluids of different genesis. T-lymphocytes of healthy donors showed only in 60% positive findings of acid alpha-naphthylacetate esterase. Positive reactions appear as dot-like reaction products in cytoplasm of lymphocytes. The application of this method on pleural fluids is possible, without technical problems. We found differences in the percentage of positive-reacting lymphocytes in pleural fluids between tuberculosis (= 78% T-cell esterase positive lymphocytes) and tumors (= 65% T-cell esterase positive lymphocytes). Mesotheliomas exhibit very low positive findings (= 32% T-cell esterase positive lymphocytes) in contrast to carcinomas. In this way, acid alpha-naphthylacetate esterase may be helpful in some cases as a diagnostic aid.

The simultaneous autoradiography and the appearance of acid alpha-naphthylacetat esterase as a cytochemical T cell marker show that only in few T cells of the tonsil a DNA-synthesis takes place. Their labelling index is lower than a power of ten compared to B cells.

Different methods for determination of T-lymphocytes in human peripheral blood were compared: rosetting with sheep erythrocytes (SRBC), AET-treated SRBC, immunofluorescence using a monoclonal antibody against T cells (BL-T2), complement dependent cytolysis with polyclonal antisera against thymocytes, cytochemical demonstration of unspecific acid alpha-naphthyl-acetate esterase (ANAE), and electrophoretic mobility using a cell electrophoresis system (PARMOQUANT 2). Depending on the method, mean values between 70 and 79% T cells among separated mononuclear cells (MNC) were found. All paired observations were subjected to statistical analysis using rank correlation and U-test. From this analysis it is concluded that rosetting with SRBC, immunofluorescence using the monoclonal T-cell antibody and cytochemical reactivity for ANAE are favored methods for determining the T cell content of human MNC. However, the monoclonal antibody BL-T2 and the ANAE are not generally applicable because both markers were also found on malignant B-lymphocytes (B-CLL).

In a female patient with histiologically ensured histiocytosis X, cell cultures modified according to NEZELOF were performed in methylcellulosis and Eagle's medium. From the 5th to the 15th day of incubation the cells were daily observed natively through a normal microscope and taken for histochemical examinations and for checking their capacity of phagocytosis. Two cell types could be distinguished: spindle-shaped fibroblasts as well as round cells of changing form and size, the alpha-naphthylacetate esterase response of which proved to be positive. The round cells can partly be regarded as HK cells, whereas giant cells could be predominantly observed during the second week. A phagocytosis of iron chips and human erythrocytes loaded with antibodies could be revealed in both types. HK cells represented mature macrophages, from which giant cells developed under in vitro conditions. There is still not explanation as to the local and immunological factors characterizing the clinical picture of histiocytosis X.

The enzyme naphthol-AS-D-chloracetate esterase (NASDCE) was demonstrated histochemically in mast cells of tissues of laboratory animals and men under normal and pathological conditions using the method after Leder (1964). Investigating the possible action of different fixations as Bouin's and Carnoy's fluid as formaldehyde also cryostate sections were studied for comparison. After tissue fixation the activity of the NASDCE is localized strongly in the granules of mast cells. The type of fixation is without influence on the histochemical reaction and localization. Differences concerning the investigated species could not be demonstrated. In benign neoplasias of mast cells such as diffuse (urticaria pigmentosa) and localized cutaneous mastocytosis as well as benign systemic mastocytosis with skin involvement (Sagher) the granule bound reaction is very intensive. The NASDCE can be recommended as a marker for the microscopical localization of mast cells, especially in the histological diagnosis of their benign and malignant neoplasias.

In the last three years, five permanently in-vitro growing cell cultures with malignant properties were established from tumour material of patients with histologically confirmed Hodgkin's disease. Four cell lines have been maintained in culture. L 428 had identical characteristics in every respect with Hodgkin and Sternberg-Reed cells, tested in-vivo on biopsy tissue. The other lines--L 538, L 540 and L 591 - had certain characteristics of Hodgkin and Sternberg-Reed cells with a number of markers, but were not fully congruent. All lines reacted with a heterologous antiserum against L 428, which selectively cross-reacted with Hodgkin and Sternberg-Reed cells in fresh biopsies. Two sublines, L 428 KS and L 428 KSA, were established from L 428 by modifying the culture medium. Tests on L 428 KS cells with conventional methods and with monoclonal antibodies demonstrated that this line carried antigens of myeloid cells; however, it could not be definitely placed into any haematopoetic line. Conditioned medium of L 428 and its sublines showed CSF activity (colony-stimulating factor) and suppression of cell-mediated cytolysis.

Enzyme-cytophotometrical investigations allow evaluation of enzyme activity within single, morphologically classified cells. Special problems like development of blood cells, bone marrow cells or of macrophages may thus be studied. Knowledge about the kinetics of granulopoiesis allows a better understanding of altered functional characteristics of abnormally developed blood cells. Enzyme cytochemical microdensitometry promises to be a useful tool in haematological cytology.

Ehrlich ascites tumor cells in peritoneal exudate of mice show a more regular round shape, greater and multiple nucleoli and more abundant and dark cytoplasma in comparison with cells of the same tumor transplanted in the limb. In solid tumor cells activity of naphthol-AS-acetate esterase is significantly higher than in exudate cells which demonstrate higher activity of PAS reaction. Other cytochemical reactions show no significant differences between the two cell forms. It seems that exudate cells are biologically more active than cells from the solid tumor.