Alpha-naphthyl Acetate Esterase Research Articles

Enzyme Histochemistry of Normal and Neoplastic Transitional Epithelium

The authors utilized the technic of plastic embedding with enzyme histochemistry for the evaluation of enzymatic expression by epithelium of the lower urinary tract in humans. Alpha-naphthyl acetate esterase and acid phosphatase generally were expressed by normal and neoplastic urothelium. Expression of 5'-nucleotidase and ATPase was more restricted. Alkaline phosphatase, prominent in the transitional cells of lower mammalian species, generally was not present in human urothelium. Enzyme histochemistry has not been applied generally to the study of disease of the lower urinary tract, but this study suggests it may be of value in understanding the biology of this tissue and be of potential use in histopathologic diagnosis of diseases of this region.

Primary tissue culture of spontaneously regressing flat warts. In vitro attack by mononuclear cells against wart-derived epidermal cells.

Although tumors may be resolved due to host immune response, it is difficult to obtain direct evidence of this in man. Numerous flat warts, human papilloma virus type 3-induced papillomas, disappear systemically and simultaneously after showing inflammatory changes. Histologically, there is a dense cellular infiltration composed of lymphocytes and mononuclear phagocytes as identified by alpha-naphthyl acetate esterase staining in situ, the former being predominant in most cases. The primary tissue culture of such inflamed flat warts from ten cases revealed a proliferation of wart-derived keratinocytes as is the case with ordinary flat warts. However, in nine of the ten cases, massive mononuclear cells, most of which were T-lymphocytes, migrated out of the explants and began to attack these keratinocytes, inducing degenerative changes. These findings indicate that cell-mediated tumor cell destruction rather than antiviral reaction induces systemic spontaneous regression of multiple papillomas in man.

A case of T-cell chronic lymphocytic leukemia with an unusual phenotype and central nervous system involvement

A case of T-cell chronic lymphocytic leukemia is reported. The leukemic cells had the morphologic features of medium-sized, mature-looking lymphocytes, and had an affinity for the central nervous system. Cytochemically, they were positive for alpha-naphthyl acetate esterase and acid phosphatase. They formed E-rosettes (E+) and reacted with OKT11 but not with OKT3/Leu-4, OKT4/Leu-3, OKT8/Leu-2, or OKM1, and did not possess IgG-Fc receptors (Fc gamma R). Functionally, they did not respond to phytohemagglutinin or concanavalin A, were not natural killer cells or antibody-dependent as well as alloantigen-reactive killer cells. Furthermore, they did not possess a helper or suppressor T-cell function for immunoglobulin synthesis. Results of immunologic studies suggest that the leukemic cells were derived from a normal counterpart of a lymphocyte subset present as a minor component of the peripheral blood, namely an E+, OKT3-, OKM1-, Fc gamma R- subset, the function of which is not yet identified.

Lymphoid crisis with T‐cell phenotypes in a patient with Philadelphia chromosome negative chronic myeloid leukaemia

A case of Philadelphia (Ph1) chromosome negative chronic myeloid leukaemia (CML) developed lymphoid crisis. Immunological marker studies disclosed that the lymphoid cells were sheep erythrocyte-rosetting-, Leu-1+, Leu-5+, OKT-4+, OKT-8+, common ALL antigen-, HLA-DR-, cytoplasmic and surface immunoglobulin-, MAS 036C(antithymocyte)+ (after in vitro culture) and terminal deoxynucleotidyl transferase-, indicating T-cell phenotypes, probably of common thymocytes. Cytochemical staining also demonstrated immature T-cell characters: dot-positivity for acid phosphatase and beta-glucuronidase, and negative for acid alpha-naphthyl acetate esterase. All bone marrow metaphases exhibited normal karyotypes. Our observation suggests that the neoplastic features of a common stem cell for myeloid and lymphoid cell lines are very similar in Ph1 positive and negative CMLs, and that the stem cell can differentiate towards T-lineage.

Acid hydrolase activities in B cell chronic lymphocytic leukaemia lymphocytes: Correlation of cytochemical reactions with immunological phenotype

The cytochemical reactions of 5 acid hydrolases, alpha-naphthyl acetate esterase (ANAE), acid phosphatase (AP), beta-glucuronidase, beta-glucosaminidase and dipeptidylaminopeptidase IV (DAP IV) were investigated in lymphocytes from 30 patients with B cell chronic lymphocytic leukaemia (B-CLL). Based on ANAE and AP reactivities, 4 cytochemically distinctive subgroups were identified: Group 1: AP and ANAE less than 50% positive lymphocytes (5 cases); Group 2: AP greater than 50%, ANAE less than 50% positive lymphocytes (11 cases); Group 3: AP less than 50%, ANAE greater than 50% positive lymphocytes (7 cases); Group 4: AP and ANAE greater than 50% positive lymphocytes (7 cases). beta-Glucuronidase displayed similar patterns of reactivity to AP. beta-Glucosaminidase activity was observed in the majority of lymphocytes in most patients, whereas DAP IV activity was present in less than 20% of lymphoid cells. The study failed to establish any relationship between cytochemical grouping and patients' clinical status, peripheral lymphocyte counts, E or mouse rosette values, light or heavy chain cellular immunoglobulin (Ig) class. Attempts to correlate acid hydrolase and Ig heavy chain isotype expression, putative markers of B cell maturation, were unsuccessful and indicate that within the narrow spectrum of B cell differentiation seen in B-CLL these characteristics are unrelated.

Isolation of plasma membrane from human blood monocytes. Subcellular fractionation and marker distribution.

The isolation of plasma membrane from human peripheral blood monocytes is described. Monocytes were isolated by centrifugal elutriation, to eliminate an adherence step, thus minimizing functional and surface antigenic alterations to the cells. Monocytes were surface-labelled with a radiolabelled monoclonal antibody, 125I-WVH-1, and then disrupted by nitrogen cavitation. Membranes were separated according to equilibrium buoyant density by isopycnic centrifugation on a sucrose gradient. The subcellular membranes were localized using marker enzymes for the plasma membrane, 5'-nucleotidase and leucine 2-naphthylamidase (leucine aminopeptidase), and for intracellular membranes: galactosyltransferase (Golgi), arylsulfatase C (endoplasmic reticulum), monoamine oxidase (mitochondria), catalase (peroxisomes), beta-hexosaminidase and beta-glucuronidase (lysosomal vesicles) and lactate dehydrogenase (cytosol). The monoclonal antibody 125I-WVH-1 was shown to label the plasma membrane, as judged by known markers, and represents a highly specific trace label, applicable to the use of plasma membrane as an immunogen for monoclonal antibody production. The NAD-splitting enzyme, NAD+ nucleosidase, was detected and its presence on the plasma membrane was demonstrated. The subcellular localization of non-specific esterase in human mononuclear phagocytes is controversial. No evidence was found for alpha-naphthyl acetate esterase activity on the plasma membrane or in lysosomal vesicles. However, a membrane-bound esterase in fractions with properties similar to the smooth endoplasmic reticulum was detected.

Identification and quantification of monocytes in bovine mononuclear cells.

To identify and quantify bovine monocytes in peripheral blood mononuclear cells (MNCs), five procedures were used: neutral red-, latex- and yeast- ingestion methods, peroxidase staining and alpha naphthyl acetate esterase (ANAE) staining. The yeast ingestion method proved to be a simple and suitable procedure for discrimination between monocytes and lymphocytes. ANAE staining was also useful for identifying monocytes. Simultaneous application of phagocytosis of yeast and ANAE staining was the most reliable procedure for the identification of bovine monocytes.

Suppression of murine natural killer cell activity by adherent cells from aging mice

Natural killer (NK) cell activity declines with age in mice. The purpose of this study was to investigate the effect of peritoneal and splenic adherent cells from young and old mice on NK activity to determine whether adherent cell suppressor function might contribute to this decline. Peritoneal adherent cells from old mice suppressed NK activity of young splenic non-adherent indicator cells more than peritoneal cells from young mice. Splenic adherent cells from old but not from young mice also suppressed this activity. That (1) the suppressive activity of the adherent cell populations was not affected by treatment with anti-Thy-1 plus complement, and that (2) the adherent cell population contained 77-92% cells positive for alpha-naphthyl acetate esterase activity, suggests that the active adherent suppressor cell may be a macrophage. Therefore, the age-related decline in NK activity in mice can be explained, in part, by an increase in adherent cell suppressor function.

Enzyme Histochemistry of Normal and Neoplastic Transitional Epithelium

The authors utilized the technic of plastic embedding with enzyme histochemistry for the evaluation of enzymatic expression by epithelium of the lower urinary tract in humans. Alpha-naphthyl acetate esterase and acid phosphatase generally were expressed by normal and neoplastic urothelium. Expression of 5'-nucleotidase and ATPase was more restricted. Alkaline phosphatase, prominent in the transitional cells of lower mammalian species, generally was not present in human urothelium. Enzyme histochemistry has not been applied generally to the study of disease of the lower urinary tract, but this study suggests it may be of value in understanding the biology of this tissue and be of potential use in histopathologic diagnosis of diseases of this region.

Cytochemical and immunological characteristics of acute monocytic leukaemia.

Immunological and cytochemical findings are presented from 12 cases of morphologically unequivocal acute monocytic leukaemia (AMoL). The results indicate considerable heterogeneity and three main non-morphological subgroups were identified. The blast cells from half the patients were positive for the presence of both cytoplasmic alpha naphthyl acetate esterase (ANAE) and monocyte-associated membrane determinants whereas the cells from three cases lacked detectable monocytic antigens despite the presence of strong cytochemical ANAE activity. A further three cases expressed monocytic antigens but were cytochemically unreactive for ANAE. These cytochemical results, which were extended by electrophoretic studies of ANAE isoenzymes, suggest that the absence of significant cytoplasmic ANAE activity does not preclude the diagnosis of AMoL and that serum lysozyme estimations may be of value in the recognition of immunocytochemically-atypical cases.

A study of certain functional parameters of monocytes from patients with multiple myeloma: comparison with monocytes from healthy individuals.

Selected functions (i.e. phagocytosis and chemotaxis of circulating blood monocytes were studied on cells obtained from 20 patients with untreated multiple myeloma (MM) and the results were compared with those obtained on cells from 60 healthy persons. The mean number of circulating monocytes was only slightly increased over the normal value although a marked monocytosis was demonstrated in all of the 4 patients with M-components of the lambda light chain type. Plasma cells isolated from peripheral blood and from bone marrow of patients with MM were found to possess a strong alpha-naphthyl acetate esterase activity. No differences were observed in the phagocytic activity of monocytes from patients compared with those of the controls, whereas the chemotactic responsiveness of the monocytes from the patients was slightly increased.

Cytochemical and electrophoretic characterisation of alpha naphthyl acetate esterases (ANAE) in acute myeloblastic leukaemia.

The alpha naphthyl acetate esterase (ANAE) cytochemical staining patterns were examined in 40 cases of acute myeloblastic leukaemia (AML: FAB groups M1 and M2) classified by morphological and immunological criteria. The blast cells in most cases (62%) were ANAE-negative with the remainder showing diffuse, granular or focal reactions of varying intensity. The nature of cytoplasmic ANAE enzymes was further characterised in 20 cases by isoelectrophoretic analysis of ANAE isoenzymes. The results suggest that the presence of significant cytoplasmic ANAE reactivity in leukaemic myeloblasts is not due to the presence of monocyte-associated isoenzymes, in otherwise well-defined myeloblasts, but may reflect abnormally increased synthesis or atypical localisation of normally-occurring ANAE isoenzymes. In particular, the results of this study indicate the lack of discriminatory value of ANAE cytochemistry in the differentiation of AML from other acute leukaemias of non-monocytic type.

Characteristics of the Strongly Ia‐Positive Cells in Rat Liver

The expression of MHC class II antigens (Ia) on rat liver non-hepatocytic cells were analysed by the Staphylococcus aureus rosette method using monoclonal antiserum to the common part of the class II molecules. Of the non-hepatocytic liver cell population, the passenger lymphocytes and monocytes were strongly anti-Ia binding but the most reactive were large mononuclear cells, morphologically macrophages. The characteristics of the strongly Ia-positive cells of rat liver were tested with different 'macrophage markers'. Of the macrophage-like cells, 55% were Ia-positive in the Staph. aureus rosette 84% had intracytoplasmic lysozyme, 100% of them were positive for alpha-naphthyl acetate esterase (ANAE) reaction, and 92% had Fc-receptors on the cell surface. As functional tests of macrophages, phagocytosis and adhesion were used: 79% of the cells were able to phagocytose antibody-coated human red cells and 56% of the cells adhered on glass. As most of the tested cells were positive for the markers the results demonstrate that the most antigenic component of rat liver has the characteristics of macrophages and thus the strongly Ia-positive cells are probably liver tissue macrophages (Kupffer cells).

Angiofollicular lymph node hyperplasia (Castleman's disease): an immunohistochemical and enzyme-histochemical study of the hyaline-vascular form of lesion.

Tissues from five cases of angiofollicular lymph node hyperplasia have been studied. All had the histological structure of the hyaline-vascular type of lesion; large numbers of very compact lymphoid follicles were distributed evenly throughout a highly vascular tissue. The follicles were characterized by their small size, a vascular poorly cellular and frequently hyalinized centre, and a 'tight' concentric mantle of small lymphocytes arranged in layers producing an 'onion-skin' appearance. The interfollicular tissue was characterized by the large numbers of small vessels mainly hyalinized capillaries and a few high endothelial venules and the presence of variable numbers of lymphocytes, plasma cells, immunocytes and immunoblasts. The immunoperoxidase method demonstrated polytypic cytoplasmic immunoglobulin in the small numbers of centroblasts and plasma cells within the follicle centres and in the plasma cells and immunocytes in the interfollicular tissue. Large numbers of suppressor T cells were present in the interfollicular areas and only scattered helper T cells were seen within the lymphocyte mantles. A strong reaction for factor VIII-related antigen was seen in the endothelium of the interfollicular high endothelial venules but only a weak reaction in the vessels in the follicle centres. A concentric distribution pattern of the dendritic reticulum cells was seen with the metalophil impregnation method of Marshall and with the enzyme histochemical methods for acid alpha-naphthyl acetate esterase and 5'-nucleotidase. This pattern differs from the zonal distribution of these cells seen in reactive lymphoid follicles. The nature and possible pathogenesis of AFLNH are discussed and contrasted with reactive hyperplasia.

Dipeptidylaminopeptidase IV activity in normal and leukemic T-cell subpopulations.

Dipeptidylaminopeptidase IV (DAP IV) cytochemical reactivity was investigated in monoclonal antibody defined T-lymphocyte subpopulations from normal blood and in cells from a series of T-cell leukemias of defined immunologic phenotype. A combined monoclonal antibody/immunocolloidal gold technic, which enabled simultaneous visualisation of immunogold label and DAP IV reactivity, was used to study enzyme reactivity in normal T-cell subpopulations. Single or several discrete granules of DAP IV reaction product were observed in 72% of OKT3+ and OKT4+ cells, whereas a significantly (P less than 0.01) lower percentage of OKT8+ cells (41%) displayed positivity; B-cells were invariably DAP IV negative. In the T-cell leukemias, DAP IV reactivity was strongest in T-lymphoblastic lymphoma and T-prolymphocytic leukemia cells. In contrast, DAP IV activity was absent or expressed in a minority of cells of the more immature T-acute lymphoblastic leukemia. The enzyme reaction also was negative in lymphocytes from other mature T-cell leukemias: T-chronic lymphocytic leukemia (OKT8+), adult T-cell lymphoma-leukemia, and Sezary syndrome (both OKT4+). DAP IV expression did not parallel that of acid phosphatase or alpha-naphthyl acetate esterase in leukemic T-lymphoid cells except in T-lymphoblastic lymphoma and T-prolymphocytic leukemia, where a strong reaction with the three hydrolytic enzymes was observed.

Human papilloma virus (HPV) antigens and local immunologic reactivity in oral squamous cell tumors and hyperplasias.

A series of 191 oral mucosal tumors including those with suspected HPV (Human Papilloma Virus) etiology; squamous cell papilloma (SQP), condyloma acuminatum (CA), focal epithelial hyperplasia (FEH), as well as those regarded as unrelated to HPV; fibrous hyperplasia (FH), papillary hyperplasia (PH), and true fibroma (TF), were analyzed for HPV structural proteins (by indirect immunoperoxidase IP-PAP technique), for the presence of epithelial dysplasia, and for the cellular composition (B and T lymphocytes, mononuclear phagocytes, (MPS cells] of their local inflammatory cell infiltrates using ANAE-(acid alpha-naphthyl acetate esterase) stain. HPV structural proteins were disclosed in 85% of FEH, in 75% of CA, and in 41% of SQP. These three lesions significantly differed from PH and FH with regard to the intensity and cellular composition of the local infiltrates, being most intense and B cell predominated in the latter two. Mild dysplasia was found in 20% of both CA and SQP lesions, the former also showing moderate dysplasia in 12% of cases. The HPV antigen positivity was negatively correlated with dysplasia in CA and SQP, the intensity of the infiltrate showing positive association with dysplasia. The results are discussed in terms of HPV etiology of CA, SQP and FEH, of the host immunologic reactivity against these lesions, as well as of the possible role of HPV in human squamous cell carcinogenesis.

Enzyme histochemical characterization of mononuclear cells in human dental periapical chronic inflammatory lesions.

Cell infiltrates in sections of paraffin-embedded tissue of 36 human periapical inflammatory lesions (15 granulomas and 21 cysts) were examined by staining for acid alpha-naphthyl acetate esterase (ANAE). Thirty-four of the lesions showed ANAE reactive T lymphocytes, plasma cells and cells of the monocyte/macrophage cell line. Large granular lymphocytes (NK cells) could not be identified with certainty among the ANAE positive cells. Two specimens were free of ANAE reactive cells. Differential cell counts on sections from 15 lesions (7 granulomas and 8 cysts) showed that T lymphocytes were the predominating ANAE reactive cells (23%), followed by plasma cells (15%) and monocytes/macrophages (11%). 51% of the inflammatory cells were ANAE negative. There was no marked difference between the granulomas and the cysts. Inhibition with NaF (10-100 mM) gave a gradual reduction in number of ANAE positive cells, some macrophages being the only cells resistant to 100 mM NaF. The results suggest that both humoral and cellular immune reactions are likely to occur in periapical inflammatory lesions.

Immunohistochemical and Electron Microscopic Characterization of the Cellular Infiltrate in Alopecia (Areata, Totalis, and Universalis)

The inflammatory cell infiltrates in scalp skin of 35 patients, 20 with alopecia areata (AA), 7 with totalis, and 8 with universalis were characterized with the ANAE (alpha-naphthylacetate esterase) marker, monoclonal antibodies, and electron microscopy. As demonstrated by the ANAE staining, no clear difference in the main lymphocyte subclasses (T and B cells) or macrophages was seen between the different types of alopecia or as compared to control patients' scalp skin. However, T lymphocytes and macrophages were seen most frequently and in greater numbers perivascularly and infiltrating the hair bulb in those cases of AA where active hair loss took place. Using monoclonal OKT (OKT-3, -4, and -8) antibodies and the avidin-biotin immunoperoxidase method on frozen sections, a concentration of OKT-8 reactive cells (suppressor/cytotoxic T cells) was seen peribulbarly and invading the hair infundibulum. The cells affecting the hair infundibulum were further studied by electron microscopy. They could be classified into three main types: small lymphocytes (60%), macrophages (30%) and cells closely resembling large granular lymphocytes (LGL) (10%). LGL have previously been considered to be human natural killer (HNK) cells. Thus the hair follicle seems to be the target for the cellular immune response in alopecia. Whether HNK cells participate in the destruction of hair bulbs remains to be investigated.

Changes in the proportion and absolute number of T lymphocytes in piglets from birth until after weaning and in adults

The proportion of T lymphocytes was measured on cytocentrifuged leucocytes prepared directly from whole blood after rosetting with sheep red blood cells in the presence of dextran. This method gave similar results to those obtained with conventional mononuclear preparations assessed in a haemocytometer but had the advantages of simplicity, the small volume of blood required and the ease with which absolute T lymphocyte counts could be estimated. The proportion of T lymphocytes did not change between birth and 12 days after weaning and did not differ from adults. However, the number of T lymphocytes at birth was significantly lower than in adults and rose thereafter to values significantly higher than in adults by 12 days after weaning. A comparison between one-day-old piglets and adults revealed that there was no significant difference in the proportion of either T or B lymphocytes between adults and neonates. However, there was some indication that neonates were deficient in the proportion of T lymphocytes capable of binding sheep red blood cells with high affinity. After examining the alpha-naphthyl acetate esterase activity of rosetted lymphocytes it was concluded that this enzyme was not a reliable marker for pig T lymphocytes.

Fractionation and further characterization of granulocytic and monocytic alpha-naphthyl acetate (ANAE) esterases.

Following characterization of myeloid nonspecific esterases by isoelectric focusing (IEF), two main groups of alpha-naphthyl acetate (ANAE) esterase isoenzymes were defined and fractionated from cytoplasmic extracts by chromato focusing techniques according to differences in their isoelectric points (pI). The first of these ANAE enzyme groups was common to leukocytes of both granulocytic and monocytic lineage, while the other, which characteristically comprised a group of isoenzymes within the pI range 5.5-6.1, was specifically associated with monocytic differentiation. The properties of the two purified ANAE enzyme fractions were compared by inhibition (heat and sodium fluoride) and further electrophoretic studies, and the results discussed in relation to the cytochemical characterization of these enzymes as markers of specific myeloid cell differentiation.