Naphthol AS-D Chloroacetate Esterase Activity Research Articles

ACTIVITY OF LEUKOCYTE ENZYMES IN AMERICAN MINK (NEOVISON VISON): GENOTYPIC AND AGE-RELATED DIFFERENCES

The activity and localization of enzymes in blood leukocytes were examined in dark brown (+/+), silver-blue (p/p) and sapphire (a/a p/p) minks during several periods of postnatal development. This study was performed by morphometric and cytochemical methods using light microscopy and image analysis software “VidеoTest». Significant genotypic differences were determined in the distribution of peroxidase, naphthol AS-D chloroacetate esterase (NASDCE) and alkaline phosphatase (AP) in leukocytes. Sapphire mink (a/a) are characterized by the lowest reproductive potential and increased postnatal mortality among the studied genotypes. Leukocytes of sapphire mink showed a significant increase in the size of and a change in the number of peroxidase- and esterase-positive structures, as well as the presence of phosphatase-negative areas in the cytoplasm. Apparently, these structures are abnormal granules and indicate the disturbance of granulogenesis. Mink of all the three genotypes demonstrated similar age-related changes of peroxidase and alkaline phosphatase activities. The lowest peroxidase activity was observed in early postnatal development, whereas the highest – at the age of 120 days. The increase in alkaline phosphatase activity occurred during the phase of active growth – on the 60th day of postnatal ontogeny. The pattern of age-related changes in NASDCE activity depended on the mink’s genotype. Sapphire mink had the highest level of NASDCE activity at the age of 4 days and the lowest – at 180 days, whereas both dark brown and silver-blue mink at the same age (180 days) had the highest values of NASDCE activity. The postnatal dynamics of the enzymes’ activity in the three genotypes apparently reflects the specific features of metabolism in leukocytes in different periods. The cytochemical features of peripheral blood leukocytes in sapphire mink suggest the presence of a morphofunctional cellular defect, which is the cause of the animals’ low viability.

Distinguishing between proliferating nodal lymphoid blasts in chronic myelogenous leukemia and non‐Hodgkin lymphoma: Report of three cases and detection of a bcr/abl fusion signal by single‐cell analysis

Lymph node biopsies were analyzed from three patients with chronic myelogenous leukemia (CML) showing nodal blast proliferation. Immunohistochemically, the blasts from all three patients had an immature marker profile with a T-blast population (cCD3+, CD4-, CD7+, CD8-, CD99+, terminal deoxynucleotidyl transferase +) and a hematopoietic progenitor cell marker (CD34). In two patients, the blasts also expressed myeloid lineage specificity (naphthol AS-D chloroacetate esterase activity and myeloperoxidase positivity). However, it was difficult to distinguish between blast proliferation in CML and non-Hodgkin lymphoma from these immunohistopathological findings alone. Subsequently, bcr gene rearrangement and bcr/abl mRNA expression were detected by Southern blot and reverse transcription-polymerase chain reaction analysis of the lymph nodes. Fluorescence in situ hybridization (FISH) analysis of lymph node touch smears also disclosed bcr/abl gene fusion signals in the blasts of all patients, confirming that the blasts were derived from Philadelphia chromosome-positive CML. Accurate discrimination between the proliferating nodal blasts of CML and non-Hodgkin lymphoma is essential for determining subsequent therapy. FISH analysis of bcr/abl in single-cell blast preparations is an efficient tool that allows rapid, accurate cytopathological diagnosis of extramedullary blast-phase CML and its discrimination from non-Hodgkin lymphoma.

Hematologic reference intervals for koi (Cyprinus carpio), including blood cell morphology, cytochemistry, and ultrastructure.

Hematologic data are used routinely in the health care of humans and domestic mammals. Similar data for fish are largely fragmentary or have not been collected. The primary purpose of this study was to determine hematologic reference intervals for koi, an ornamental strain of the common carp (Cyprinus carpio). Secondarily, the morphology, cytochemical reactions, and ultrastructure of koi blood cells were characterized. A CBC was performed manually on heparin-anticoagulated blood specimens using Natt and Herrick's diluent and a Neubauer-ruled hemacytometer. Leukocyte differential counts were done on Wright-Leishman- and Diff-Quik-stained blood smears. Cytochemical reactions of koi leukocytes were determined using commercial kits. Transmission electron microscopy was performed to characterize the ultrastructural features of koi blood cells. Hematologic reference intervals were established for healthy koi for PCV (30-34%), hemoglobin concentration (6.3-7.6 g/dL), RBC count (1.7-1.9 X 10(6)/ microL), WBC count (19.8-28.1 X 10(3)/ microL), RBC indices, and differential leukocyte counts. Lymphocytes were the predominant leukocyte (accounting for up to 80% of all leukocytes), whereas eosinophils were rare. Basophils were positive with PAS staining. Naphthol AS-D chloroacetate esterase activity was observed only in eosinophils. alpha-Naphthyl butyrate esterase and beta-glucuronidase activities were positive in monocytes. Some lymphocytes were reactive for alpha-naphthyl butyrate esterase and acid phosphatase activity. Ultrastructurally, leukocytes, erythrocytes, and thrombocytes were identified on the basis of cytoplasmic organelles and granule appearance. Hematologic reference intervals and knowledge of the cytochemical reactions and ultrastructural characteristics of koi leukocytes will help standardize hematologic studies in this species.

Brain injury after survived gunshot to the head: reactive alterations at sites remote from the missile track

Gunshot wounds to the brain usually lead to acute respiratory arrest or death after a brief survival period, even in cases involving only slight direct tissue damage. It can be assumed therefore that the damage extends beyond the zone of recognizable destruction and hemorrhages. To determine the true extent of the tissue injury resulting from gunshot wounds to the brain, we carried out microscopic investigations for reactive changes (emigration of leukocytes and macrophages, axonal expression of β-amyloid precursor protein (β-APP) in 10 cases of gunshot wound to the narrow channel of the brain with survival times >2 h. Demonstration of leukocytes expressing naphthol AS-D chloroacetate esterase activity in the brain tissue at the border of the missile track established the vitality of the gunshot effect. The presence of macrophages (CD68-epitope) allowed demarcation of a 1–2 mm wide necrotic zone around the permanent cavity. Within this zone and beyond, β-APP showed an initial increase followed by a decline in the number of injured axons. Three types of β-APP positive staining could be differentiated. In the immediate vicinity of the missile track β-APP positive neurons were present at a distance of 2–4 mm from the margin of the permanent cavity (type 1) as a result of primary injured neuronal tissue by the gunshot itself. At longer distances from the narrow channel and the permanent cavity single β-APP positive axons or axon fragments and two additional types were found; type 2 shows a parallel, wave-like arrangement of the damaged fibers, which suggests that the injury was produced by mechanical acceleration of the brain tissue created by the energy the projectile expended within the brain; irregular aggregation of β-APP positive axons or axon fragments within a local edema represents type 3, which may be attributed to secondary ischemia or edema.

Hypericin induces both differentiation and apoptosis in human promyelocytic leukemia HL-60 cells.

Hypericin is a unique photosensitizing plant pigment and has been separately reported to induce differentiation and apoptosis in neoplastic cells. In this study, we examined the relationship between activities to induce differentiation and apoptosis in human promyelocytic leukemia HL-60 cells, at a concentration range of 0.15 to 0.2 microM. When treated with hypericin, the cell ratio reducible of nitroblue tetrazolium was significantly increased and the cell size was enlarged by flow cytometry analysis. Hypericin also significantly increased the ratio of the cells, which were of positive alpha-naphthyl acetate esterase activity and phagocytic activity, whereas it hardly influenced the naphthol AS-D chloroacetate esterase activity in the cells, as well as 1 alpha, 25(OH)2D3 (10 nM). In addition, hypericin increased hypodiploid nuclei and caused a nucleosomal ladder. These results indicate that hypericin induces both differentiation toward monocyte/macrophage lineage and apoptosis in HL-60 cells.

Induction of differentiation of the human histocytic lymphoma cell line U-937 by hypericin.

Hypericin, a photosensitizing plant pigment, was found to be a potent inducer of differentiation of human myeloid leukemia U-937 cells. At a concentration of 0.2 microM, hypericin exhibited 50% growth inhibition. An effect on cell differentiation by hypericin was assessed by its ability to induce phagocytosis of latex particles, and to reduce nitroblue tetrazolium (NBT). Approximately 51% of 0.2 microM hypericin-treated cells were stained with NBT and 63% showed phagocytic activity. In order to establish whether hypericin induces differentiation of U-937 cells to macrophage or granulocyte, esterase activities and cell sizes were measured. When U-937 cells were treated with 0.2 microM and 0.15 microM of hypericin, the alpha-naphthyl acetate esterase activity was increased by 38.4% and 48.1%, respectively, but naphthol AS-D chloroacetate esterase activity was not influenced. The size of hypericin-treated cells in terms of cell mass was larger than that observed in untreated cells as determined by flow cytometry. Protein kinase C (PKC) inhibitor, NA-382, decreased the NBT reducing activity of hypericin, whereas a cAMP-dependent protein kinase A (PKA) inhibitor, H-89, did not show any influence on the differentiation. These results indicate that hypericin triggers differentiation toward monocyte/macrophage lineage by PKC stimulation.

An Unusual Form of Chronic Myeloproliferative Disorder Aleukemic Basophilic Leukemia

A 52-year-old Japanese man manifested various clinical signs and symptoms such as vomiting, high fever, dyspnea, cough, sweating, palpitation, eosinophilic leukocytosis and hepatosplenomegaly. These histamine-related clinical manifestations showed a dramatic response to steroid therapy. After 10 months of hospitalization, he suddenly succumbed to candidal septicemia at the end of the third cycle of steroid therapy. Autopsy revealed neoplastic proliferation of immature basophils in various internal organs without involvement of the skin. The neoplastic cells, positive immunohistochemically for leukocyte common antigen, possessed lobulated nuclei and weakly metachromatic cytoplasmic granules, predominantly of the basophil type, which exhibited weak naphthol ASD-chloroacetate esterase activity. Mast cell-type granules were also observed ultrastructurally. The neoplastic infiltration was associated with fibrosis in the liver, spleen and bone marrow and with extramedullary hematopoiesis in the liver, spleen, lymph nodes and perihypophyseal tissue. The bone marrow showed uneven and multifocal involvement. Despite the lack of leukemic manifestations and the results of chromosomal analysis, the most suitable diagnosis was aleukemic basophilic leukemia within the category of chronic myeloproliferative disorder. Kinship of this neoplasia to systemic mastocytosis is discussed.

Myeloblastoma of the brain

Two patients developed intraparenchymal myeloblastomas (granulocytic sarcomas) of the brain. One patient with acute myelogenous leukemia in hematological remission had multiple myeloblastomas in the cerebrum and thalamus. The second patient, who had Philadelphia-chromosome-positive chronic myelogenous leukemia, developed a solitary lesion of the cerebrum without systemic evidence of blastic transformation of leukemia. By light microscopy, each biopsy specimen revealed a tendency for the malignant cells to appear in perivascular locations, and the presence of cytoplasmic naphthol ASD chloroacetate esterase activity in some cells. Ultrastructural analysis demonstrated the presence of primary granule-like lysosomal structures in these cells, confirming their granulocytic origin. The lack of continuity of the myeloblastomas with the dura mater and the perivascular clusters of blast cells suggest that the pathogenesis of these lesions in the present cases could best be accounted for the direct, hematogenous spread of mitotically competent leukemic cells to brain parenchyma.

Inhibition of non-specific leukocyte esterase activity. Absence of monocyte esterase activity due to phosphoric and thiophosphoric acid ester intoxication

In vitro evaluation of the effect of five insecticidal phosphoric and 11 thiophosphoric acid esters on different, non-specific human leukocytes esterases indicated that most of the organic phosphor compounds studied inhibited the activity of neutral α-naphthylacetate esterase, α-naphthylbutyryl esterase, and naphthol AS acetate esterase, i.e. the monocyte esterases. The extent of inhibition was dose dependent; the inhibiting dose being identical for the various non-specific esterases. Reactivation with Obidoxim was not successful. Monocyte esterase activity in a human survivor of E 605 intoxication was detectable only after serum acetylcholinesterase had returned to normal levels. The organic phosphor compound studied, however, inhibited neither acid α-naphthylacetate esterase nor naphthol AS-D chloroacetate esterase activity.

Circulating micromegakaryocytes preceding leukemia in three dogs exposed to 2.5 R/day gamma radiation.

The presence of micromegakaryocytes in the blood and bone marrow of humans during the preleukemic phase of acute and chronic myelogenous leukemia and myelomonocytic leukemia is well documented [I, 4, 5, 7, 8, 10, 15), and it has been suggested that it is the single most typical abnormality suggesting a subsequent course towards overt leukemia (6). Three adult purebred beagles that died with myelogenous leukemia during continuous whole-body exposure to 2.5 roentgens/22-hour day of 6OCO gamma irradiation had micromegakaryocytes and megakaryoblasts in the peripheral blood three to ten weeks before leukemic myeloblasts were observed in buffy coat preparations. The cells of interest were 6 to 30 JLm in diameter, mono-, or binucleated, with round or oval nuclei, indistinct nucleoli, and they had a high nuclear/cytoplasmic ratio (fig. I). Many cells had cytoplasmic blebs (fig. 2). Cytochemical characterization revealed strong positive staining for both acetylcholinesterase, and a-naphthyl acetate esterase, as well as abundant glycogen with the periodic acid-Schiff stain. The cells were negative with Sudan black, and no detectable myeloperoxidase or naphthol AS-D chloroacetate esterase activity was seen. Other hematologic abnormalities observed during this preleukemic period included progressive refractory anemia, occasional nucleated red blood cells, anisopoikilocytosis, monocytosis, giant platelets, and a left shift to metamyelocytes. Over the same period, bone marrow preparations from iliac crest aspirates and rib biopsies revealed granulocytic hyperplasia with a moderate left shift but without an excess of myeloblasts. There was also erythroid depletion, and increased numbers of mono-, bi-, and multinucleated megakaryocytes (fig. 3). Ultrastructural characterization clearly indicated the cells to be micromegakaryocytes and megakaryoblasts. Even in the most immature cells, the cytoplasm contained both dense serotonin granules and the characteristic a (bull's-eye) granules, indicating their megakaryocytic lineage (fig. 4). Although the majority of micromegakaryocytes showed dysgenesis of the demarcation membrane system, the cytoplasm did contain smooth, membranous vesicular sacs of a rudimentary demarcation membrane system (fig. 4) and in a few cells there were flattened, laminar cisternae of the demarcation membrane system with peripheral formation of dystrophic (i.e., agranular, giant) platelets. Terminally, the dogs were severely anemic (0.94, 1.10, and 1.70 X 10 red blood cells/ul), and thrombocytopenic (5.0, 7.5, and 90.0 X 10 Total leukocyte counts were 11.5, 14.8, and 57.1 X 10/ JLI, respectively. The terminal peripheral blood differential leukocyte count showed a marked left shift (17%myeloblasts) in only one dog. However, bone marrow imprints obtained at necropsy from sternum, rib, and femur showed, in all dogs, a granulocytic hyperplasia with maturation defects and myeloblast counts of 21.0, 11.0, and 35.5% of the total granulocytic series. In all dogs there was a paucity of erythroid elements and in only one dog were there a few remaining megakaryocytes.

Hydrolysis of a chymotrypsin substrate and of naphthol AS-D chloroacetate by human leukocyte granules.

The subcellular distribution has been investigated of two esterases from human neutrophil granulocytes obtained from patients with chronic myelocytic leukemia. One esterase hydrolyzed the typical chymotrypsin substrate N-acetyl-L-tyrosine ethyl ester, the other hydrolyzed naphthol AS-D chloroacetate, a substance which is used in cytochemistry for the demonstration of esterase activity. Both enzymes could be recovered almost exclusively in the granule fraction and were optimally active at about pH 7.4. After lysis of the granules by hypoosmotic shock 73% of the naphthol AS-D chloroacetate esterase activity could be dissolved in buffered 0.15M sodium chloride (pH 7.0) in contrast to only 17% of the N-acetyl-L-tyrosine ethyl esterase activity. The soluble lysate contained 59% of the total granule protein. It was subjected to acrylamide-gel electrophoresis at acid pH and multiple bands of naphthol AS-D chloroacetate esterase activity were demonstrated on the gels. Acrylamide-gel electrophoresis of the insoluble proteins and the soluble lysate of the granule fraction was carried out after dissolution of the proteins in phenol-acetic acid-urea and the resulting electrophoretic patterns were compared. Hydrolysis of naphthol AS-D chloroacetate by the granule fraction was compared to hydrolysis of the same substrate effected by the pure proteases chymotrypsin, trypsin, and pancreatopeptidase E.