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Patients suffering from lymphogranulomatosis were studied with respect to cellular immune deficiencies. For this purpose, mononuclear cells from venous blood were separated and subjected to analysis of lymphocyte markers. T-lymphocytes were enumerated by means of the sheep erythrocyte (SE) rosette test. T cell subpopulations were determined using enzyme cytochemical staining for dipeptidyl peptidase IV (DP IV) and unspecific acid alphanaphthylacetate esterase (ANAE). In 18 patients with M. Hodgkin a significant reduction in the T lymphocyte count in peripheral blood was found. This T cell defect is due to a selective decrease in the TM-subpopulation as identified by enzyme cytochemical markers DP IV and ANAE (focal reaction). From these results it is concluded that patients with lymphogranulomatosis have characteristic abnormalities in the immune system in the sense of a disturbed equilibrium of immune regulatory cells.

The method for purification of monocytes using adherence to gelatin coated glass surfaces described by Chien et al. was optimized by drastic shortening the incubation time and modifying the culture media. After one adherence step we obtained monocytes with a purity of 73-78% and a recovery of 53%. Thiol-protein-disulfide-oxidoreductase (TPO), a new enzyme marker of monocytes, was found to be also valid for monocytes obtained by adherence methods. Comparing the number of TPO-containing monocytes with other markers (alpha-naphthylacetate esterase, peroxidase, phagocytosis of latex particles, acridine orange fluorescence, antigens detected by the monoclonal BL-M/G antibody) almost identical values were found.

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).

Cholesteryl ester storage disease is a rare disorder if cholesterol metabolism characterized by excessive hepatic storage of cholesteryl esters. The underlying defect probably is decreased activity of alpha-naphtyl-acetatesterase, a lysosomal acid lipase. The leading symptom in the early stage is a pronounced enlargement of the liver without subjective complaints or other changes in the physical status. Diagnosis can be established by liver biopsy, which will yield characteristic findings, and by exclusion of other storage disease. Histologic examination of the biopsy specimen will show lipid droplets in hepatic parenchymal cells, vacuolated Kupffer cells and focal accumulation of foamy cells. Biochemical analysis of the biopsy specimen will show an increased content of cholesteryl esters. Investigation of media of culture fibroblasts will show decreased activity of lysosomal alpha-naphtyl-acetat-esterase. In addition adrenal calcification has to be ruled out, in order to exclude wolman's disease. The prognosis of the disease will depend upon the degree of liver insufficiency, since lympho-histiocytic infiltration as a secondary phenomenon may lead to septal fibrosis and cirrhosis in children or adolescents in most cases. A case report is given of a 13-year old girl suffering from this disease.

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.

Blood was taken from the femoral vein of 17 autopsied cadavers with different diagnoses and postmortal intervals (10-120 h). The granulocytes and lymphocytes were isolated with routine methods. The cell suspensions were subject to morphologic, enzyme-cytochemical (naphthol AS-D chloroacetate esterase) and electrophoretic (PGM1, PGM3, GOTM, PEPA, MEM, FUCA) investigations. The following results were obtained: Erythrocyte-free cell suspensions are only found during short postmortal intervals; Isolation of pure lymphocytes from cadaver blood using the described method is only possible very early in the postmortal interval (within 10 h); The isolated granulocytes contain an impressively high percentage of eosinophilic granulocytes; The percentage of naphthol AS-D-chloroacetate esterase-positive granulocytes is considerably lower in stored, conserved blood than in blood smears; Identification of the above mentioned enzymes from isolated granulocytes from all cadavers is possible by electrophoresis.

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.

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.

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.

The diagnostic significance of acid non-specific alpha-naphthyl-acetate (ANAE) and of rosette formation of leukemic cells with sheep and mouse erythrocytes was studied in 8 patients with acute myeloic leukemia (AML), in 4 patients with acute lymphatic leukemia (ALL) and in 14 patients with chronic lymphatic leukemia (CLL). ANAE showed typical diffuse cytoplasmic activity in all cases of AML. The enzyme activity was granular in both of the lymphoid malignancies, T-ALL and B-CLL, allowing differentiation from AML but not between B- and T-leukemia cells. Rosette formation with mouse erythrocytes (ME) was diagnostic in all 14 cases of CLL and superior to labeling of surface immunoglobulin (8 of 14 cases positive). ME-rosette forming myeloblasts were detected in 2 of 4 evaluated cases of AML. Rosette formation with sheep erythrocytes (SE), including cytological evaluation of rosette-forming cells, was diagnostic in all cases of ALL (= T-ALL). In 6 of 9 patients with AML, however, rosette formation with SE was observed in a few cells, including myeloblasts with Auer rods. The occurrence of blasts in myeloic leukemia carrying lymphoid cell markers is discussed in the light of recent findings, according to which lymphoblastoid cells may arise in the course of myeloic leukemia requiring antileukemic treatment different from that of AML.

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.

Heparinized venous blood was stored under sterile conditions at different temperatures (4 C, 20 C, 37 C) for various intervals (0-7 days). After storage the granulocytes and lymphocytes were isolated with routine methods. Naphthol AS-D-chloroacetate esterase as a granulocyte marker and acid alpha-naphthyl acetate esterase as a T-lymphocyte marker were identified on smears of the washed cell suspension. Different enzymes were identified in the cell sediment with electrophoresis. Relatively pure lymphocyte suspensions were obtained within the first 24 h. After this time, however, the percentage of these mononuclear cells declined markedly. The percentage of isolated granulocytes varied slightly; there was a marked predominance of granulocytes (more than 70%) at all intervals investigated during the isolation. Cytochemical analysis of the granulocytes and lymphocytes indicated that the decrease in the percentage of enzyme-positive cells depends in each case on the duration of the storage interval. During the first 24 h, only PGM1 and GOTM could be identified in the lymphocyte suspension with horizontal starch gel electrophoresis. The enzymes PGM1, PGM3, PGI, MDH, GOTM, 6-PGD, ADA could always be identified in the granulocyte suspension; AK, FUCA, MEM could be occasionally identified; and GPT and GLO could never be identified.

A comparison was made between the cytochemical behaviour of PAH stimulated lymphocytes and that of non-stimulated lymphocytes otherwise treated in the same way. Apart from non essential differences and the clear increase in size of the stimulated cells, there was no significant difference in the behaviour of stimulated and non-stimulated lymphocytes during the various reactions (non-specific esterases, acid phosphatase, alkaline phosphatases, naphtol ASD sodiumacetate-esterases, peroxydases leucine aminopeptidases, DOPA-reaction). There was no sign of lymphocytes being transformed into monocytes of fibroblasts.

On continuation of study by KURZ and GOSLAR (1974) concerning the inhibitory effects to non specific esterases on liver and kidney, the behaviour of alpha-naphthyl-acetate-esterases on hypothalamus, neurohypophysis, adrenal and parotid glands of rats had been examined against aldehydes, organic solution-mediators and anorganic salts. It had shown a different sensitiveness, the esterases of tanycytes, nerve cells and pituicytes acted more sensitive than those of the adrenal cortex and parotid gland. The differences seen on the solution-mediators were less; on metal salts from different groups of periodical system which applied, the most intense blocking effect was found in the nerve cells. The esterases of tanycytes and the immediately extensively reacting pituicytes, as well as the adrenal cortex esterases reacted essentially less sensitive, the gland cells of parotid even almost not at all. The arguments of this behaviour are discussed and pointed out with different complexity of alpha-naphthylacetate splitting enzymes on individual organs.

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.

In the brain of the Caecilian species Chthonerpeton indistinctum the following enzymes have been demonstrated by means of histochemical techniques: acid phosphatase, alpha-naphthylacetate esterase, acetylcholin esterase. Acid phosphatase occurs in the cytoplasm of the neurons in 4 different types of localization. Its activity in the ventral parts of the brain is markedly higher than in the dorsal ones. Of particularly high activity are: the motor neurons in the tegmentum, the nucleus mesencephali trigemini, individual large neurons in the marginal zone of the grey matter of the telencephalon, which seems to be a special character of the Caecilians among the Amphibia. The ependyma exhibits local differences in respect of acid phosphatase activity. alpha-Naphthylacetate esterase marks in particular the secretory neurons of the hypothalamus, the large perikarya of the nucleus mesencephali trigemini and the motor neurons of the tegmentum. In the telencephalon the alpha-naphthylacetate esterase activity corresponds to that of acid phosphatase. Acetylcholin esterase marks--with certain restrictions--cholinergic neurons. These predominate in Chthonerpeton in the caudal parts of the brain. In the telencephalon amygdala, septal area striatum and the mitral cells are of comparatively high activity. The neurosecretory neurons of the hypothalamus are particularly rich in this enzyme. As an anurans the cholinergic fasciculus retroflexus as asymmetric. The tectum opticum is of secondary simplicity and does not exhibit a clearly recognizable stratification.

We tested how long unstained and unfixed films of blood and aspirated bone marrow could be kept before special cytochemical staining. To exclude influences other than time on the outcome of the reactions, all the material was taken from hematologically normal persons. A simple score was used to evaluate the results. These may be summarized as follows: in most cells Naphthol-AS-acetate-esterase first shows a progressive decline in reactivity after a delay of 6 weeks, the reaction becoming very weak after 10 +/- 1 weeks. This does not hold for monocytes, megakaryocytes and platelets, which maintain reactivity (and susceptibility to NaF) for more than three months. With PAS staining, a slight decrease in reactivity after 8 +/- 2 weeks could be observed, whereas all other stainings tested (POX, Sudan black B and Naphthol-AS-D-esterase) were unaffected even by a delay of up to 16 weeks.