A study of peripheral blood in hedgehogs in Turkey.

The aim of this study was to determine diameters of blood cells, differential counts of peripheral blood leukocytes, alpha-naphthyl acetate esterase (ANAE), acid phosphatase (ACP-ase) activity of some leukocyte types, and enzymatic positivity percentages of peripheral blood lymphocytes in two hedgehogs species, Hemiechinus auritus, the long-eared hedgehog, and Erinaceus concolor, the southern white-breasted hedgehog. Air-dried peripheral blood smears were stained with May-Grünwald-Giemsa stain. ANAE and ACP-ase were stained in glutaraldehyde-acetone-fixed smears. ANAE-positive lymphocytes displayed a dot-like positivity pattern characterized with 1-5 reddish brown cytoplasmic granules, whereas ACP-ase positive lymphocytes displayed a dot-like positivity pattern characterized with 1-3 pinkish cytoplasmic granules. Monocytes gave a diffuse and strong reaction while neutrophils displayed a weak positive reaction for ANAE and ACP-ase. No difference was observed in mean diameters of peripheral blood cells of these species. It was found that lymphocytes made up the majority (64.3% and 65.5%) of leukocytes, followed by neutrophils (23.9% and 23.3%), eosinophils (9.0% and 7.6%), monocytes (1.8% and 2.3%), and basophils (1.0% and 1.3%) in H. auritus and E. concolor, respectively. Mean ANAE positivity oflymphocytes was 36.6% and 51.3% and ACP-ase positivity was 32.1% and 37.5% for H. auritus and E. concolor, respectively. The ANAE positivity of lymphocytes in E. concolor was significantly (P < 0.05) higher than that of H. auritus.

Effects of paclitaxel and resveratrol on blood characteristics in rabbits.

We investigated the adverse effects of paclitaxel (PAC) on blood characteristics including the percentage of alpha naphthyl acetate esterase (ANAE)-positive peripheral blood lymphocytes, white cell count, number of neutrophil nuclear lobe and, erythrocyte diameter, and the protective effects of resveratrol (RES) on these characteristics. Male New Zealand rabbits were assigned to four groups. The control group (C) was given 40 ml normal saline, the PAC group was given 5 mg/kg PAC in 40 ml normal saline, the RES group was given 4 mg/kg RES in 40 ml normal saline, and the PAC + RES group was given 5 mg/kg PAC + 4 mg/kg RES in 40 ml normal saline. All injections were performed intravenously once/week for 8 weeks. PAC caused an increased total percentage of lymphocytes and monocytes, which was associated with neutropenia. The PAC group showed a right shift in the lobulation curve of neutrophil nuclei together with a decreased percentage of ANAE-positive lymphocytes. RES reduced the percentage of ANAE-positive lymphocytes, slightly increased the percentage of neutrophil leukocytes and reduced the total lymphocyte count. Administration of RES combined with PAC prevented, while PAC induced, neutropenia and lymphocytosis, and increased the percentage of ANAE-positive lymphocytes.

Leukocyte transmigration in inflamed liver: A role for endothelial cell-selective adhesion molecule.

BACKGROUND/AIMS: This study was designed to investigate the role of endothelial cell-selective adhesion molecule (ESAM), a recently discovered receptor expressed in endothelial tight junctions and platelets, for leukocyte migration in inflamed liver. METHODS: The role of ESAM for leukocyte migration in the liver was analyzed using ESAM-deficient mice in a model of warm hepatic ischemia-reperfusion (90min/30-360min). RESULTS: As shown by immunostaining, ESAM is expressed in sinusoids as well as in venules and is not upregulated upon I/R. Emigrated leukocytes were quantified in tissue sections. Postischemic neutrophil transmigration was significantly attenuated in ESAM-/- mice after 2h of reperfusion, whereas it was completely restored after 6h. In contrast, T-cell migration did not differ between ESAM+/+ and ESAM-/- mice. Using intravital microscopy, we demonstrate that ESAM deficiency attenuates I/R-induced vascular leakage after 30min of reperfusion. The I/R-induced elevation in AST/ALT activity, the sinusoidal perfusion failure, and the number of TUNEL-positive hepatocytes were comparable between ESAM+/+ and ESAM-/- mice. CONCLUSIONS: ESAM is expressed in the postischemic liver and mediates neutrophil but not T-cell transmigration during early reperfusion. ESAM deficiency attenuates I/R-induced vascular leakage and does not affect leukocyte adherence. Despite the effect on neutrophil migration, ESAM-deficiency does not protect from I/R-induced injury.

Effects of long term oral acrylamide administration on alpha naphthyl acetate esterase and acid phosphatase activities in the peripheral blood lymphocytes of rats.

Acrylamide is an important industrial chemical; it also is formed in starch-rich foodstuffs during baking, frying and roasting. Most acrylamide exposure occurs by ingestion of processed foods. We investigated possible immunotoxic effects of extended administration of low doses of acrylamide in rats. To do this, we measured alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (ACP-ase) activities in peripheral blood lymphocytes. Male and female weanling Wistar rats were administered 2 or 5 mg acrylamide/kg/day in drinking water for 90 days. Peripheral blood was sampled at the end of the administration period. We found ANAE staining in eosinophils and T-lymphocytes, but not in monocytes, platelets, B-lymphocytes and neutrophils. ACP-ase was found in B-lymphocytes. We found a significant reduction of the ratio of ANAE:ACP-ase in lymphocytes of the experimental animals compared to controls. We found no statistically significant differences between the doses or sexes. We found that acrylamide ingested in processed foods might affect the immune system adversely by decreasing the population of mature T- and B-lymphocytes.

Cytochemical characterization of peripheral blood cell populations of two Cyprinidae, Carassius auratus and Ctenopharyngodon idellus.

Fish are the most diverse species of all vertebrate groups, and their blood cells have shown variable characteristics in terms of morphology. Cytochemical staining for enzyme activity in blood leukocytes will help assess the immune function of fish. We characterize blood cells from crucian carp (Carassius auratus) and grass carp (Ctenopharyngodon idellus) by using a Diff-Quick stain as well as different cytochemical methods. Blood specimens obtained from crucian carp and grass carp were evaluated after cytochemical staining for acid phosphatase (ACP), alkaline phosphatase (ALP), naphthol AS chloroacetate esterase (AS-DNCE), naphthyl acetate esterase (NAE), α-naphthyl butyrate esterase (NBE), peroxidase (MPO) and periodic acid-Schiff's reaction (PAS) using commercial kits. Blood cell types were evaluated based on their morphological characteristics and the presence or absence of specific chromogen. The expression pattern of enzymes was similar between the two Cyprinidae and was also broadly consistent with other fish species. However, there were some interesting differences detected between crucian carp and grass carp, including naphthol AS chloroacetate esterase activity in monocytes, peroxidase activity and location in thrombocytes. The ACP, ALP and MPO expressions of different leukocytes of the two Cyprinidae were evaluated by Image Pro Plus and were analysed for statistical significant differences. This investigation provides basic haematology and enzyme activity analyses for crucian carp and grass carp and serves as an approach to evaluating the immune response of fish.

Protective effect of quercetin on some hematological parameters in rats exposed to cadmium.

We investigated the effects of quercetin (Q) on some hematological parameters and determined the percentage of alpha-naphthyl acetate esterase (ANAE) positive lymphocytes in rats that had been exposed to cadmium (Cd). Thirty male Wistar albino rats were divided into four groups: control (C), quercetin (Q), cadmium (Cd) and Q + Cd (CdQ). Blood samples were taken to assess erythrocytes (RBC), leukocytes (WBC), hemoglobin levels (Hb), hematocrit values (Hct), platelets (PLT), alpha-naphthyl acetate esterase (ANAE) positive lymphocytes. RBC, Hb, Hct; the number of PLT significantly decreased in the Cd group. To the contrary, these parameters were increased significantly in the CdQ group compared to the Cd group. Although we found a significant increase in total WBC count and neutrophil percentage, the number of lymphocytes decreased in the Cd group compared to the other three groups. Also, the percentage of peripheral blood ANAE positive lymphocytes decreased significantly in the Cd group (p < 0.05). Q exhibits positive effects on some hematological characteristics and the percentage of ANAE positive lymphocyte in cases of acute CD toxicity.

Hematology and enzyme histochemistry of the peripheral blood leucocytes in rock partridges (Alectoris graeca).

The purpose of this study was to determine the percentages of alpha-naphthyl acetate esterase (ANAE)-positive and acid phosphatase (ACP)-positive peripheral blood lymphocytes (PBL) and the percentages of leukocytes in the rock partridge at different ages. Blood samples obtained from the vena basilica of 18 healthy rock partridges (Alectoris graeca) at 1 d, 5 wk, and 12 wk of age were used. Mean percentages of ANAE-positive PBL for the 3 age groups were 37, 29.83, and 47.83% for 1 d, 5 wk, and 12 wk of age, respectively. Heterophils also gave ANAE-positive reactions. Mean percentages of ACP-positive PBL for the 3 age groups were 70, 81, and 86.1% for 1 d, 5 wk, and 12 wk of age, respectively. Although monocytes showed a diffuse granular staining pattern, heterophils displayed a weak positive reaction for ACP. Thrombocytes showed a small granular staining pattern. This study contributes by broadening the hematological research on avian species and provides a guideline for identifying blood cells in the rock partridge.

[Cytochemical activity of cells of the lymphocytic-macrophageal system in patients with chronic acquired toxoplasmosis].

The peripheral blood lymphocytic activities of acid phosphatase (AP) and acid nonspecific esterase (ANE) (alpha-naphthylacetate esterase) were determined in 45 patients with chronic acquired toxoplasmosis on an exacerbation. There was a considerable increase in the activity of AP in the white blood cells, which being more pronounced in the monocytes. The activity of ANE was moderately enhanced in the lymphocytes. The determination of the leukocytic activities of AP and ANE may be used as additional tests in the diagnosis of an exacerbation of chronic acquired toxoplasmosis and in the evaluation of the efficiency of the treatment performed.

Phorbol diester-induced macrophage differentiation of leukemic blasts from patients with human myelogenous leukemia.

Phorbol esters, including 12-O-tetradecanoylphorbol 13-acetate (TPA), induce terminal macrophagelike differentiation of cells from human acute myelogenous leukemia lines. We report that myelogenous leukemia cells obtained from patients undergo macrophagelike differentiation after exposure to TPA. The myeloid leukemic cell cultured with TPA became adherent to charged surfaces with long filamentous pseudopodia; developed positive staining for alpha-napthyl acetate esterase, increased lysozyme secretion, reduced nitroblue tetrazolium, and acquired the ability to phagocytose candida. Cells from patients with lymphocytic leukemia did not become macrophagelike when cultured with TPA.

Differential sodium fluoride sensitivity of alpha-naphthyl acetate esterase in human, bovine, canine, and murine monocytes and lymphocytes.

The sensitivity of alpha-naphthyl acetate esterase (ANAE) activity of human, bovine, canine, and murine leukocytes to low (42 micrograms/ml incubation mixture) and high (1.5 mg/ml incubation mixture) concentrations of sodium fluoride (NaF) was compared. The ANAE activity of bovine and canine monocytes was only slightly inhibited by the low concentration of NaF, whereas that of murine peritoneal macrophages was moderately inhibited and that of human monocytes was completely inhibited. Human and canine monocytic ANAE was completely inhibited by the high concentration of NaF, whereas the ANAE activity in 1% of bovine monocytes and murine peritoneal macrophages was not inhibited. The ANAE activity of human and murine lymphocytes in nonenriched venous blood samples showed no or negligible NaF sensitivity to the low concentration of NaF, whereas bovine lymphocytic ANAE showed slight and canine lymphocytic ANAE moderate NaF sensitivity. The ANAE of murine T-lymphoma L5178Y cells was resistant to the low concentration of NaF, but the ANAE of both murine T-lymphoma L5178Y cells and murine lymphocytes was completely inhibited by the high concentration of NaF. Human, bovine, and canine lymphocytic ANAE showed marked NaF sensitivity toward the high concentration of NaF, showing 73%, 86%, and 93% inhibition, respectively. The neutrophils in murine venous blood and ascites were positive for ANAE activity, which was only slightly inhibited by the low concentration of NaF but completely inhibited by the high concentration. Results of this investigation demonstrate that the ANAE of leukocytes of various species show remarkable differences in its sensitivity to low and high concentrations of NaF, indicating that appropriate concentrations of NaF are required for distinguishing the ANAE of lymphocytes and monocytes of different species.

Demonstration of membrane-bound proteolytic activity on the surface of mononuclear leukocytes.

The existence of proteolytic enzymes bound to the surface of migrating cells has often been surmised. That such enzymes are present on mononuclear leukocytes was suggested by studies showing that serum amyloid A (SAA), the presumed precursor of amyloid protein A, is degraded in the presence of monocytes without endocytosis and with only negligible activity in the cells' supernates. Experiments using immunofluorescence were designed to support this view. It was shown that SAA binds to the cells' surface at low temperatures, whereas binding at 37 degrees C could only be demonstrated when the cells were pretreated with the serine protease inhibitor, diisopropyl-fluorophosphate (DFP) or the elastase inhibitor Ac-Ala-Ala-Pro-Val-CH2Cl. Exposure of the cells to these inhibitors before incubation with SAA at 0 degrees C permitted detection of the protein for an indefinite period of time. At 37 degrees C the DFP-treated cells polarized and eventually lost the surface-bound protein. No interiorized SAA could be demonstrated. Radioautography of cells that had been treated with 3H-DFP revealed grains on the plasma membrane and in the cytoplasm of sectioned monocytes, whereas only 8-15% of lymphocytes were labeled. In lymphocytes radioactivity was restricted to the surface membrane. Additional experiments showed that alpha-naphthyl acetate esterase activity is also located on the surface of monocytes and a subpopulation of lymphocytes. These observations have led to the conclusion that some functions of mononuclear leukocytes may be mediated by enzymes associated with the external surface of these cells.

Endotoxin-induced tissue factor in human monocytes is dependent upon protein kinase C activation.

Tissue factor (TF) is a transmembrane receptor which, in association with factors VII and VIIa, activates factor IX and X, thereby activating the coagulation protease cascades. In response to bacterial lipopolysaccharide (LPS) monocytes transcribe, synthesize and express TF on their surface. We investigated whether LPS-induced TF in human monocytes is mediated by protein kinase C (PKC) activation. The PKC agonists phorbol 12-myristate 13-acetate (PMA) and phorbol 12, 13 dibutyrate (PdBu) were both potent inducers of TF in human monocytes, whereas 4 alpha-12, 13 didecanoate (4 alpha-Pdd) had no such effect. Both LPS- and PMA-induced TF activity were inhibited, in a concentration dependent manner, by three different PKC inhibitors: H7, staurosporine and calphostin C. TF antigen determination confirmed that LPS-induced cell-surface TF protein levels decreased in parallel to TF functional activity under staurosporine treatment. Moreover, Northern blot analysis of total RNA from LPS- or PMA-stimulated monocytes showed a concentration-dependent decrease in TF mRNA levels in response to H7 and staurosporine. The decay rate of LPS-induced TF mRNA evaluated after the arrest of transcription by actinomycin D was not affected by the addition of staurosporine, suggesting that its inhibitory effect occurred at a transcriptional level. We conclude that LPS-induced production of TF and its mRNA by human monocytes are dependent on PKC activation.

Morphological and biochemical characterization of Procambarus clarki blood cells.

Morphological and cytochemical analysis of Procambarus clarki hemocytes demonstrated three cell types: hyaline, semigranular, and granular. Hyaline cells showed a higher nuclear/cytoplasmic ratio with few small electron-dense granules in the cytoplasm. Semigranular cells presented numerous round or oval eosinophilic granules (0.40-0.78 micron). Granular cell contained large eosinophilic granules (1.79-3.05 microns). Ultrastructurally, all cells showed microtubules near the borders, a poorly developed Golgi complex, and secretory-type electron-dense particles. No mitotic figures were seen. Cell monolayers showed three morphologically distinct cell types (composed of flattened and well-spread cells) depending on the presence and size of granules (hyaline, semigranular, and granular). No sex-related differences could be documented in cell features or proportions. Cytochemical studies showed that the three cell types were positive for acid phosphatase. Granular and semigranular cells were also positive for nonspecific esterase. Phenoloxidase activity was localized only in granular and semigranular hemocytes, and peroxidase activity was observed only in the granular hemocytes. These results may suggest that the semigranular and granular hemocytes participate in the prophenoloxidase system and also in phagocytic or cytotoxic function.

Alpha-naphthyl acetate esterase activity in human foetal lymphocytes.

Human foetal mononuclear cells from thymus, spleen, liver, bone marrow and peripheral blood at 8-24 weeks of gestation were examined for cytochemical evidence of acid alpha-naphthyl acetate esterase (ANAE) activity. The focal brownish-red ANAE reaction product (T cell staining pattern) was observed in counterstained cytocentrifuged cell smears in the cytoplasm. ANAE-positive lymphoid cells were first observed in the thymus at 9 weeks of gestation. A gradual increase in frequency of ANAE-positive cells in foetal thymus was observed, from about 10% at 14-15 weeks to about 20% at 22-24 weeks of gestation. By 14 weeks of foetal age, spleen and liver contained a few ANAE-positive cells and after 15 weeks of gestation consistent occurrence of ANAE-positive cells was observed in foetal bone marrow and peripheral blood. These results demonstrate that ANAE-positive lymphocytes first appear in the foetal thymus and are subsequently found in the foetal liver, spleen, bone marrow and peripheral blood.

Factors affecting acid alpha-naphthyl acetate esterase activity in peripheral blood lymphocytes.

Pre-staining conditions that affect alpha-naphthyl acetate esterase (ANAE) activity in peripheral blood lymphocytes have been evaluated. The effects of different fixatives, pH of fixation, pre-fixation storage time and length of fixation have been compared with regard to maximal preservation of ANAE activity in peripheral T-cells.

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.

Acid phosphatase and alpha-naphthyl acetate esterase in neoplastic and non-neoplastic lymphocytes. A statistical analysis.

Acid phosphatase and alpha-naphthyl acetate esterase reaction patterns were evaluated in lymphocytes from patients with a variety of neoplastic and non-neoplastic conditions: leukemia, 59; NHL, 53; and reactive follicular hyperplasia, 23. Fifteen individuals with normal peripheral blood were also studied. For both enzymes, statistical analysis showed a strong correlation between a globular reaction pattern and T lymphocytic origin in both non-neoplastic lymph nodes and normal peripheral blood specimens (P less than 0.0001). A similarly strong correlation was found between a granular acid phosphatase pattern and T lymphocytic origin in cell isolated from non-neoplastic lymph nodes (P less than 0.0001) but not in those obtained from normal peripheral blood where this pattern was observed with equal frequency in B, T, and "null" lymphocytes (P = 0.415). A granular alpha-naphthyl acetate esterase pattern was correlated with non-T lymphocytes from normal peripheral blood (P less than 0.0001), but was observed with equal frequency in B, T, and "null" lymphocytes fron non-neoplastic lymph nodes (P = 0.76). In the eight T cell neoplasias studied, a globular pattern was evident in the majority of cells for both enzymes. In the majority of the B cell neoplasias, however, a granular pattern was observed for both enzymes.

Nonspecific esterase staining patterns in acute monocytic leukemia.

Staining patterns for nonspecific esterase (NSE) were analyzed retrospectively in 14 cases of acute monocytic leukemia (M5) using alpha-naphthyl acetate as substrate. An attempt was made to correlate variability in NSE activity with clinical findings. Two staining patterns were distinguished: (1) diffuse fine granular cytoplasmic staining, and (2) dense focal staining superimposed on a weak positive cytoplasmic background. The 14 cases were divided into two groups according to their different staining qualities: Group 1 (6) diffuse and Group 2 (8) focal. All six cases of Group 1 were typical hypercellular acute leukemias. Group 2 contained four cases of hypercellular acute leukemia and four cases of hypocellular acute leukemia. All the cases of hypocellular leukemia showed the pattern of focal NSE activity. No difference in the clinical presentation or course of the disease between Groups 1 and 2 was found.

Effect of cytostatics administration in rats on leukocyte cytochemical markers.

The influence of cytostatic drugs (busulphan, chlorambucil, prednisone, 6-mercaptopurine, L-asparaginase) on the activity of alkaline and acid phosphatase, alpha-naphtolacetate esterase and the content of glycogen and lipids in leukocytes of peripheral blood in healthy rats was investigated. No analogy was reported between the changes of cytochemical reactions in healthy population of leukocytes in rats as compared with the changes (observed in preceding papers) of cytochemical reactions in leukocytes of patients with leukemia under the influence of the same cytostatic drugs.

Influence of chemotherapy on some cytochemical reactions in patients with acute leukemia.

The influence of cytostatic drugs (L-asparaginase, vincristine, 6-mercaptopurine, amethopterine, prednisone) on the activity of alkaline and acid phosphatase, alpha-naphtol-acetate esterase, the content of glycogen and lipids in leukocytes of peripheral blood in patients with acute leukemia was investigated. Under the influence of anti-leukemic drugs some cytochemical reactions typically changed in different forms of acute leukemia showed tendency to normalization being sometimes more distinctive than leukocytosis or even than white blood picture. In patients who did not show any improvement during the treatment the disturbances of cytochemical reactions intensified or, sometimes, remaining unchanged. The repetition of examination of cytochemical reactions changing distinctively in the chemotherapy may simplify the treatment control by better estimation of its efficiency and give some prognostic hints.