Naphthyl Acetate Research Articles

Prognostic significance of acid alpha naphthyl acetate esterase and acid phosphatase in childhood acute lymphoblastic leukemia

We studied the prognostic value of the enzymes acid alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (AP) in 89 children with acute lymphoblastic leukemia (ALL). Follow-up data were available for 61 out of cases of non T- non B-ALL, which were treated in different hospitals according to the same protocols. Sex, age, initial white blood cell count (WBC) and number of high risk patients (WBC above 25 × 10 9/1 were comparable between enzyme-positive and -negative cases. The probabilities of complete continuous remission (CCR) were virtually identical in the AP + and AP − group. For the ANAE + group the probability of CCR was lower than for the ANAE - group, but this difference was not statistically significant (0.10 > p > 0.05). Within the common-ALL group ( n = 32), no difference was found in probability of CCR between the AP + and AP − group but ANAE + cases had a significantly lower probability of CCR than ANAE − cases. This study is a contribution to the view that the cytochemical profile of ALL cells may have prognostic value.

Quantitative enzyme determination; a parameter for leukemic cell differentiation

In acute myeloid leukemia the leukemic cells are thought to be blocked in their normal differentiation. The stage of differentiation is the basis for the FAB classification. Induction of cell differentiation is a new and promising development in the treatment of some myeloproliferative diseases. The criteria for cell maturation and differentiation are almost all based on the morphology of the leukemic cells. In this study we tried to specify the maturation stage of the leukemic cells by quantitative enzyme analysis. In the HL60 (promyelocytic) cell line cells we determined the following enzymes: myeloperoxidase; alpha naphthyl acetate esterase; alpha naphthyl butyrate esterase and lactate dehydrogenase. The enzyme profiles obtained after culturing the cells in the presence of the differentiation inducing agents 1 : 25 dihydroxy vitamin D3 and DMSO were compared with several cytochemical and functional parameters. The results obtained in this study show that quantitative enzyme analysis is a useful tool in the study of myeloid or monocytic differentiation.

Use of Surface Marker Analysis to Predict Outcome of Adult Acute Myeloblasts Leukemia

In order to investigate the clinical significance of surface antigen analysis in acute myeloblastic leukemia (AML), the blasts from 196 patients with AML were analyzed prospectively with a panel of 16 monoclonal antibodies. The antibodies were selected to identify differentiation-associated antigens of either the myeloid lineage (MY9, PM-81, AML-2-23, MY7, MCS-1, MY8, Mo1, MY1, MY4, Mo2), T cell lineage (T101, T11), B cell lineage (B1, B4) or multiple lineages [J5 (CALLA), HLA-DR]. Independent morphological review and classification by French-American-British (FAB) criteria was performed in 161 of the 196 cases. One or more myeloid surface antigens were detected on the blasts of 195 cases, while B and T cell markers were detected on 0% to 2% of cases. When both blood and marrow samples were studied on the same patient, very few differences were noted between the antigenic profiles of the paired specimens. The frequency of expression of individual myeloid antigens ranged from 91% (PM-81) to 29% (Mo2). Expression of individual antigens was found to correlate significantly with several clinical parameters including FAB classification, cytochemical staining for alpha naphthyl acetate esterase, leukocyte count, and the presence of extramedullary disease at presentation. Two myeloid antigens (MY4 and MY7) predicted for a low rate of complete remission (CR) to standard induction chemotherapy. MY4+ cases (37% of the total population) had a CR rate of 53%, while M4- cases had a CR rate of 69% (P = .03). MY7+ cases (57% of the total population) had a CR rate of 55% while MY7- cases had a CR rate of 73% (P = .01). Neither MY4 nor MY7 antigen expression was correlated with patient age. Paired combinations of antigens were also examined. The [MY4- MY7-] phenotype was exhibited by 32% of all cases and was associated with an 82% CR rate while the CR rate of all other cases was 54% (P = .001). The expression of three antigens (HLA-DR, MY8, Mo1) was associated with a decreased continuous complete remission (P less than .05, median follow-up time of 19 months). Expression of MY8 antigen was also associated with decreased survival (P = .03). These results confirm earlier reports of antigenic heterogeneity in AML, and indicate that immunologically defined subgroups of AML patients which are of potential clinical significance can be identified.

Selected Enzyme Histochemical Techniques Facilitated by the Microwave Oven

AbstractEnzyme procedures may require 60 minutes or more reaction time at room temperature. The microwave oven was used to produce a substrate solution internal temperature of 38°C, which allowed the demonstration of enzyme activity of acid phosphatase, alkaline phosphatase, alpha naphthyl acetate esterase, naphthol AS-D chloracetate esterase, and acetyl cholinesterase within a greatly reduced period. The procedures can be done on frozen sections and cold processed glycol methacrylate embedded tissues.

Use of surface marker analysis to predict outcome of adult acute myeloblastic leukemia

In order to investigate the clinical significance of surface antigen analysis in acute myeloblastic leukemia (AML), the blasts from 196 patients with AML were analyzed prospectively with a panel of 16 monoclonal antibodies. The antibodies were selected to identify differentiation-associated antigens of either the myeloid lineage (MY9, PM-81, AML-2–23, MY7, MCS-1, MY8, Mo1, MY1, MY4, Mo2), T cell lineage (T101, T11), B cell lineage (B1, B4) or multiple lineages [J5 (CALLA), HLA-DR]. Independent morphological review and classification by French- American-British (FAB) criteria was performed in 161 of the 196 cases. One or more myeloid surface antigens were detected on the blasts of 195 cases, while B and T cell markers were detected on 0% to 2% of cases. When both blood and marrow samples were studied on the same patient, very few differences were noted between the antigenic profiles of the paired specimens. The frequency of expression of individual myeloid antigens ranged from 91% (PM-81) to 29% (Mo2). Expression of individual antigens was found to correlate significantly with several clinical parameters including FAB classification, cytochemical staining for alpha naphthyl acetate esterase, leukocyte count, and the presence of extramedullary disease at presentation. Two myeloid antigens (MY4 and MY7) predicted for a low rate of complete remission (CR) to standard induction chemotherapy. MY4+ cases (37% of the total population) had a CR rate of 53%, while M4- cases had a CR rate of 69% (P = .03). MY7+ cases (57% of the total population) had a CR rate of 55% while MY7- cases had a CR rate of 73% (P = .01). Neither MY4 nor MY7 antigen expression was correlated with patient age. Paired combinations of antigens were also examined. The [MY4- MY7-] phenotype was exhibited by 32% of all cases and was associated with an 82% CR rate while the CR rate of all other cases was 54% (P = .001). The expression of three antigens (HLA-DR, MY8, Mo1) was associated with a decreased continuous complete remission (P less than .05, median follow-up time of 19 months). Expression of MY8 antigen was also associated with decreased survival (P = .03). These results confirm earlier reports of antigenic heterogeneity in AML, and indicate that immunologically defined subgroups of AML patients which are of potential clinical significance can be identified.

Coexistent chlamydial infections related to natural history of human papillomavirus lesions in uterine cervix.

To assess the role of Chlamydia trachomatis in the development of cervical intraepithelial neoplasia (CIN) and to evaluate possible synergism between chlamydiae and human papillomavirus (HPV) in this process, 418 women who had been prospectively followed up for cervical HPV infections at our clinic since 1981 were tested for chlamydiae. At each visit the patients were examined by colposcopy, and other investigations, such as Papanicolaou (Pap) smears, punch biopsies, urethral, and cervical swabs, were undertaken as indicated. In biopsy specimens the cytopathic changes of HPV, concomitant CIN, and the local immunocompetent cell infiltrates were analysed. The latter were measured and further identified using an alpha naphthyl acetate esterase (ANAE) technique to define B cells, macrophages, and T cells and using monoclonal antibodies to define T cell subsets, NK (natural killer cells), and Langerhans cells. Chlamydial isolation (4.1% in the cervix, and 3.6% in the urethra) did not positively correlate with the degree of cytological atypia in PAP smears or with the degree of CIN lesions associated with HPV. Chlamydial cervicitis did not affect the ANAE definable cell composition of the immunocompetent cell infiltrates in HPV lesions, or that of the immunocompetent cell subsets, including the ratios of T helper to T suppressor cells and the numbers of NK cells. Chlamydial infection did not alter the natural history of HPV lesions, of which 30% regressed, 53% persisted, and 17% progressed during follow up. The present results do not provide evidence to substantiate the hypothesis that chlamydiae and HPV might act synergistically in cervical carcinogenesis, or the view that C trachomatis may be a major aetiological agent of CIN lesions. Chlamydiae and HPV are covariables of sexual behaviour, and their concomitant appearance in sexually promiscuous women is best explained by this fact. As we do not have more direct evidence for the oncogenic potential of C trachomatis (as we have of HPV), it seems reasonable to consider that this agent is not a major cause of CIN, but rather a sexually transmitted agent commonly found in women with CIN because of their promiscuous sexual behaviour.

Differentiation of bone marrow cells from myelodysplastic patients in the presence of 1,25 dihydroxyvitamin D3 or 13-cis retinoic acid.

The separate effects of vitamin D3 (1,25(OH)2D3) and 13-cis retinoic acid on the differentiation in liquid culture of marrow cells from seven patients with myelodysplastic syndrome (MDS) were studied. Following incubation with 1,25(OH)2D3, an increasing number of myeloid cells acquired the morphological appearance of mature monocyte-macrophages and reacted positively to fluoride-sensitive naphthyl acetate esterase and specifically bound My4 monoclonal antibody (McAb). Incubation of bone marrow cells with 13-cis retinoic acid enhanced the number of cells with the morphological appearance of metamyelocytes and mature granulocytes as well as those that reacted positively with AS-D naphthol chloroacetate esterase. The results suggest that the differentiation pattern of myeloid precursor cells from MDS patients can be modulated by 1,25(OH)2D3 and 13-cis retinoic acid.

Cytochemical reactions in cells from leukemic dogs.

Leukemic cells from 17 dogs with spontaneous leukemia were stained with leukocyte alkaline phosphatase, alpha naphthyl acetate esterase with and without fluoride, peroxidase, and periodic acid-Schiff. Cytochemistry was necessary for identification or confirmation of leukemic cell type in most dogs and resulted in changing the light microscopic morphologic diagnosis in eight of 17 dogs. Leukemic cell types diagnosed were myelomonocytic leukemia in seven dogs, monocytic leukemia in five dogs, lymphocytic leukemia in four dogs, and myelocytic leukemia in one dog.

Cytochemical analysis of peripheral blood mononuclear cells following allogeneic bone marrow transplantation: correlation of hydrolase expression with graft-versus-host disease

Peripheral blood mononuclear cells of 21 patients undergoing allogeneic bone marrow transplantation (BMT) were monitored post-BMT for immunologic markers (E rosettes OKT3, OKT8, DR, and BT5/9, a monoclonal antibody which stains helper T cells), cytochemical markers (acid phosphatase [AP], beta-glucuronidase [BGLU], and acid alpha naphthyl acetate esterase [ANAE] ), and morphology. The cytochemical T score, was obtained from typical AP, BGLU, and ANAE reactivity. The same was done for the cytochemical non-T score and macrophage (Mo) score. All patients received cyclosporine A (CyA) for graft-v-host disease (GvHD) prophylaxis. In univariate analysis there was no significant correlation between the proportion of E rosettes, OKT3-, OKT8-, DR-, and BT5/9-positive cells, and GvHD. The first three showed instead a positive correlation with time from transplant: E rosettes (P = .02), OKT3 (P = .01), and OKT8 (P = .003). In contrast, a significant negative correlation was found in univariate analysis, between the cytochemical T score and GvHD (P = .0001), and a positive correlation between non-T score and GvHD (P = .0008), as well as between the Mo score and GvHD (P = .03). There was no influence of time from transplant on the T (P = .8), non-T (P = .8), or Mo score (P = .4). In multivariate analysis comparing E rosettes, OKT3, T score, non-T score, GvHD, and time from BMT, the only variable associated with GvHD was the T score (P less than .05). These results suggest that T cell activation during GvHD is associated with a loss of hydrolase expression in T cells, but does not imply relevant modifications of immunologic surface markers. In addition, lysosomal enzymes appear early (before day 10) after transplantation, indicating that T cells at this stage are well differentiated.

Cytochemical analysis of peripheral blood mononuclear cells following allogeneic bone marrow transplantation: correlation of hydrolase expression with graft-versus-host disease

AbstractPeripheral blood mononuclear cells of 21 patients undergoing allogeneic bone marrow transplantation (BMT) were monitored post-BMT for immunologic markers (E rosettes OKT3, OKT8, DR, and BT5/9, a monoclonal antibody which stains helper T cells), cytochemical markers (acid phosphatase [AP], beta-glucuronidase [BGLU], and acid alpha naphthyl acetate esterase [ANAE] ), and morphology. The cytochemical T score, was obtained from typical AP, BGLU, and ANAE reactivity. The same was done for the cytochemical non-T score and macrophage (Mo) score. All patients received cyclosporine A (CyA) for graft-v-host disease (GvHD) prophylaxis. In univariate analysis there was no significant correlation between the proportion of E rosettes, OKT3-, OKT8-, DR-, and BT5/9-positive cells, and GvHD. The first three showed instead a positive correlation with time from transplant: E rosettes (P = .02), OKT3 (P = .01), and OKT8 (P = .003). In contrast, a significant negative correlation was found in univariate analysis, between the cytochemical T score and GvHD (P = .0001), and a positive correlation between non-T score and GvHD (P = .0008), as well as between the Mo score and GvHD (P = .03). There was no influence of time from transplant on the T (P = .8), non-T (P = .8), or Mo score (P = .4). In multivariate analysis comparing E rosettes, OKT3, T score, non-T score, GvHD, and time from BMT, the only variable associated with GvHD was the T score (P less than .05). These results suggest that T cell activation during GvHD is associated with a loss of hydrolase expression in T cells, but does not imply relevant modifications of immunologic surface markers. In addition, lysosomal enzymes appear early (before day 10) after transplantation, indicating that T cells at this stage are well differentiated.

Genetics of an esterase associated with resistance to organophosphorus insecticides in the sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae)

AbstractElectrophoresis was used to characterize the esterases in Lucilia cuprina (Wiedemann). Of the 16 esterase bands visualized, only one was associated with resistance to organophosphorus insecticides. In laboratory reference strains, the esterase E3 was consistently found in both susceptible and heterozygously resistant flies, but was absent from homozygously resistant flies. It was postulated that resistant flies possessed a non-staining form of E3, designated E3null. Genetic analyses mapped the locus for E3null to a position in the same region as the gene for organophosphorus resistance. No recombinants between the genes for resistance and E3null were detected. In field populations of L. cuprina from several areas in Australia, a close association was found between the frequency of E (0·97) and the proportion of flies resistant to organophosphorus insecticide (0·967). This association suggests that E3null represents the product of a major resistance gene in terms of the ‘mutant ali-esterase’ theory. E3 as found in susceptible blowflies, represents the ‘original’ ali-esterase, the gene for which mutated to one coding for E3null. This mutant enzyme can hydrolyse organophosphates more efficiently than E3 but has lost the ability to hydrolyse 1- and 2-naphthyl acetate (the substrates used to visualize the esterases after electrophoresis).

Cytochemical, immunological and ANAE-isoenzyme studies in acute myelomonocytic leukaemia: a study of 39 cases.

This study reports the cytochemical, electrophoretic and immunological characteristics of blasts cells from 39 cases of acute myelomonocytic leukaemia (M4). The results indicate considerable cytochemical heterogeneity, particularly with respect to esterase (alpha naphthyl acetate and chloroacetate) activities and suggest that an increased serum lysozyme concentration is a more consistent feature. Investigations with a range of monoclonal antibodies also revealed some differences in expression of monocyte-associated determinants although it is considered that immunological assessments are more consistent than cytochemistry in the detection of monocytic blast cell components. Analysis of ANAE isoenzymes by isoelectric focusing was found to be of particular value in cases where interpretation of ANAE cytochemistry was difficult.

Induction of Hydrolases by Allelochemicals and Host Plants in Fall Armyworm (Lepidoptera: Noctuidae) Larvae

The influence of allelochemicals and host plants on two hydrolases, α -naphthyl acetate ( α -NA) esterase and styrene oxide hydrolase, was investigated in the fall armyworm, Spodoptera frugiperda (J. E. Smith). The α -NA esterase was induced by a range of allelochemicals. Celery, potato, and parsley were also active in inducing the esterase, whereas a number of other host plants had no significant effect. Styrene oxide hydrolase was moderately induced by indole 3-carbinol, peppermint oil, and phenobarbital, but none of the host plants showed any inductive effect.

Magnetic isotope and external magnetic field effects upon the photo-Fries rearrangement of 1-naphthyl acetate

The reaction mechanisms of the photo-Fries rearrangement of 1-naphthyl acetate has been studied by means of steady-state photolysis and laser flash photolysis. A radical pair consisting of the 1-naphthoxyl and acetyl radicals is concluded to be a reaction intermediate. The yield of an in-cage product (2-acetyl-1-naphthol) exhibits a positive external magnetic field effect for the ester labeled by magnetically active /sup 13/C, but no effect for the normal /sup 12/C ester. The magnetic field effect observed for the labeled ester is quantitatively or semiquantitatively explained in terms of the radical-pair mechanism by considering hyperfine coupling between magnetically active nuclei (/sup 1/H and /sup 13/C) and an unpaired electron in the acetyl radical. The in-cage product is formed through the singlet radical pair. 26 references, 5 figures, 3 tables.

Combined Enzyme Method for the Simultaneous Demonstration of Muscle Fiber Type and Neuromuscular Junctions

A method is described for distinguishing type I from type II muscle fibers and visualizing their motor endplates. Muscle biopsies from the region of the neuromuscular junction are snap frozen and sectioned on a cryostat. The sections are first incubated in a substrate containing beta nicotinamide adenine dinucleotide, reduced form, and nitroblue tetrazolium, then in a substrate containing alpha naphthyl acetate and hexazotized new feuchsin. This procedure takes only thirty-five minutes to do, no cholinesterase inhibitors are used, and both fiber type and neuromuscular junction are demonstrated simultaneously. It can be used routinely to help diagnose neuromuscular disorders and for myoneural research, in general.

Combined Enzyme Method for the Simultaneous Demonstration of Muscle Fiber Type and Neuromuscular Junctions

A method is described for distinguishing type I from type II muscle fibers and visualizing their motor endplates. Muscle biopsies from the region of the neuromuscular junction are snap frozen and sectioned on a cryostat. The sections are first incubated in a substrate containing beta nicotinamide adenine dinucleotide, reduced form, and nitroblue tetrazolium, then in a substrate containing alpha naphthyl acetate and hexazotized new feuchsin. This procedure takes only thirty-five minutes to do, no cholinesterase inhibitors are used, and both fiber type and neuromuscular junction are demonstrated simultaneously. It can be used routinely to help diagnose neuromuscular disorders and for myoneural research, in general.

Enzyme histochemical comparison of biomphalaria glabrata amebocytes with human granuloma macrophages.

In fresh water snails, amebocytes are the principal cells that react to parasitic infection. Ultrastructurally, amebocytes resemble mammalian macrophages. To clarify the relationship between amebocytes and macrophages, we compared the histochemical staining for seven enzymes in Biomphalaria glabrata snail amebocytes, both in the amebocyte-producing organ (APO) and in the encapsulation reaction formed around parasite sporocysts with the staining in macrophages from the lymph nodes of patients with sarcoid or tuberculosis. Snails were infected with Echinostoma paraensei and Schistosoma mansoni miracidia. APOs and ventricular tissue with encapsulated parasites were fixed and embedded in glycol methacrylate monomer. Hardened blocks were sectioned at 2 micron and stained for alkaline phosphatase, acid phosphatase, alpha-naphthyl acetate esterase (ANAE), ATPase, peroxidase, 5'nucleotidase, and chloroacetate esterase. The amebocyte-producing organ contained cells that were positive for acid phosphatase, ANAE, and ATPase. Amebocytes in the capsules formed around echinostome sporocysts showed stronger staining for the same three enzymes. Capsules did not form around schistosome sporocysts, but the connective tissue around them contained numerous amebocytes that were also positive for these three enzymes. The amebocyte enzyme histochemistry resembled that in human granuloma macrophages, but differed from that in neutrophils. The increased expression of enzymes in amebocytes involved in the encapsulation reaction as compared to those in the APO was reminiscent of the alterations observed when human monocytes convert to tissue macrophages. These studies support the hypothesis that the amebocyte is an "invertebrate macrophage."

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.

Effect of substrate concentration, product concentration, and peptides on the in vitro hydrolysis of model ester prodrugs by corneal esterases.

The corneal epithelial fraction of albino rabbits with butyrylcholinesterase-like activity, which was purified by gel filtration chromatography, was studied with respect to its susceptibility to substrate and product inhibition using a pH-stat method. 1- and 2-Naphthyl acetates were used as model ester prodrugs. It was found that the hydrolytic rate of 1- and 2-naphthyl acetates at 1.2 mM was only 53% and 42% of that at 0.06 mM, respectively, suggesting that increasing the dose of an ester prodrug does not necessarily result in a proportional increase in its hydrolytic rate. Product inhibition (by 1- and 2-naphthol) was evident only at a product concentration at least equal to the substrate concentration, suggesting that, at the therapeutic concentrations of an ester prodrug, product inhibition is probably insignificant. Substrate and product inhibition did not appear to occur at the enzyme's active site. Moreover, the neuropeptides enkephalins and their hydrolytic fragments were found to inhibit the hydrolysis of 1-naphthyl acetate, albeit less effectively than compounds such as 1- and 2-naphthol.

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.