Butyrate Esterase Research Articles

Proanthocyanidins from barley bran potentiate retinoic acid-induced granulocytic and sodium butyrate-induced monocytic differentiation of HL60 cells.

Polyphenol extract from barley bran (BPE) induced nitro blue tetrazolium (NBT) reducing activity and alpha-naphthyl butyrate esterase activity in HL60 human myeloid leukemia cells. Because BPE induced the biochemical markers of HL60 cell differentiation, we investigated the effects of proanthocyanidins isolated from BPE on the HL60 cell differentiation of HL60 cells. Prodelphinidin B-3, T1, T2, and T3 induced 26-40% NBT-positive cells and 22-32% alpha-naphthyl butyrate esterase-positive cells. Proanthocyanidins potentiated retinoic acid (all-trans-retinoic acid)-induced granulocytic and sodium butyrate-induced monocytic differentiation in HL60 cells.

Acute myeloid leukemia with minimal differentiation. A multiple parameter study.

Acute myeloid leukemia with minimal differentiation (AML-M0) is a recently described entity, and few large studies are available to characterize its clinical and pathologic features. We reviewed blood and bone marrow morphology, cytochemistry, immunophenotyping, and cytogenetics, as well as the clinical findings, of 17 patients with AML-M0. Most patients were male, with a median age of 62 years. Minimal background myelodysplastic features were identified in only 5 of 15 patients. Cytochemical stains for myeloperoxidase and alpha-naphthyl butyrate esterase were negative in the leukemic blasts of all specimens. Positivity for one or both myeloid-associated antigens, CD13 and CD33, was seen in all patients. Both CD34 and HLA-DR were positive in all tested cases. Lymphoid-associated antigens were identified in seven patients; these antigens were typically dim or weak in intensity. Terminal deoxynucleotidyltransferase positivity was seen in 14 of 14 tested patients; monoclonal anti-myeloperoxidase reactivity was seen in the blasts of 2 patients. Abnormal clonal karyotypes were found in 6 of 14 patients. Abnormalities of chromosomes 7 and 13 were the most common findings, most frequently manifested by monosomy 7 and trisomy 13. The median follow-up was 8 months. Eight patients died of their disease, three are alive with disease, and six are in first or second remission (including three patients treated with bone marrow transplantation). When narrowly defined, AML-M0 appears to be a homogeneous entity that affects older and predominantly male patients. It has no single karyotypic abnormality, but complex karyotypes with monosomy 7 and trisomy 13 are commonly found. Acute myeloid leukemia with minimal differentiation is relatively resistant to chemotherapy; however, bone marrow transplantation may provide a better outcome for eligible patients.

Structure-activity relationships of flavonoids and the induction of granulocytic- or monocytic-differentiation in HL60 human myeloid leukemia cells.

The flavones apigenin and luteolin strongly inhibited the growth of HL60 cells and induced morphological differentiation into granulocytes. The flavonol quercetin inhibited the cell growth and induced a differentiation marker, i.e., NBT reducing ability. However quercetin-treated cells were not morphologically differentiated into granulocytes. The chalcone phloretin weakly induced NBT reducing ability and a marker of monocytic differentiation alpha-naphthyl butyrate esterase activity in the cells. Quercetin and phloretin appeared to induce the differentiation of HL60 cells into monocytes. The proportion of alpha-naphthyl butyrate esterase-positive cells induced by genistein was less than that of the NBT-positive cells. Some of the nuclei in genistein-treated HL60 cells morphologically changed. Genistein must have induced both granulocytic and monocytic differentiation of HL60 cells. The flavonols galangin and kaempferol, which had fewer hydroxyl group(s) in the B-ring than quercetin, and the flavanone naringenin inhibited the growth but did not induce the differentiation of HL60 cells.

Enzymes Involved in Defense Functions of Hemocytes of MusselMytilus galloprovincialis

Mytilus galloprovincialishemocytes contain phosphatases, esterases, proteases, and glucosidases as revealed by a semiquantitative colorimetric method. The activity levels of some enzymes changed throughout the months of the experiment without a seasonal pattern. No differences in the enzyme levels were found between the two groups of mussels of different age (cultivated 11 and 19 months on ropes hung from the same raft). Light microscopy study of hemocyte enzymes showed that hyalinocytes and granulocytes had reaction of acid phosphatase, β-glucuronidase, α-naphthyl butyrate esterase, naphthol AS-D chloroacetate esterase, and α-naphthyl acetate esterase. Phenoloxidase and peroxidase activities were also detected in monolayers of hemocytes. Ultrastructural study revealed that the granules of hemocytes contained acid phosphatase, β-glucuronidase, nonspecific esterases, peroxidase, and NADH oxidase. Products of β-glucuronidase and peroxidase reaction were also localized in the plasma membrane. Lysozyme activity level was higher in hemocytes than in serum. Comparison between the two groups of different age showed a higher activity level of lysozyme in younger mussels.

Extracellular proteins of Cryptococcus neoformans and host antibody response.

Proteins secreted by the fungal pathogen Cryptococcus neoformans may be involved in invasion and could be useful in vaccine design. Despite the medical importance of this fungus, little is known about its extracellular proteins or the immune response to these antigens. To study C. neoformans extracellular proteins, 12 strains were metabolically radiolabeled and protein supernatants were analyzed. Both strain- and growth condition-dependent differences were observed. Enzymatic assays of filtered culture supernatants revealed butyrate esterase and caprylate esterase lipase activity for 11 of 12 strains, as well as acid phosphatase, naphthol-AS-BI-phosphohydrolase, and beta-glucosidase activities in some strains. Serum from infected rodents immunoprecipitated several secreted proteins, consistent with in vivo expression and development of an antibody response. For strain 24067, two immunodominant species, of approximately 75 and 30 kDa, were recognized. The relative intensity of the autoradiographic bands depended on the route of infection for both rats and mice. In summary, our results indicate that (i) there are multiple proteins in C. neoformans culture supernatants, (ii) there are strain differences in supernatant protein profiles, (iii) there are differences in supernatant protein profile depending on the growth conditions, (iv) there are several new extracellular and/or cell-associated enzymatic activities, and (v) antibodies to several supernatant proteins are made in the course of infection.

Correlation analysis of alveolar macrophage cytochemical parameters in smoking and pulmonary oncology.

Cytochemical examination of alveolar macrophages (AM) obtained by bronchoalveolar lavage (BAL) was performed in healthy volunteers (11 non-smokers and 11 smokers) and in 9 patients with squamous lung carcinoma (all of them smokers or ex-smokers) in order to analyze its peculiarities related to the smoking habit and to lung malignancy. Assessment of non-specific esterases: alpha-naphthyl acetate esterase (ANAE) and butyrate esterase (BUT), chloroacetate esterase (CHL), acid phosphatase (AcP), intracellular glycogen (PAS reaction), lipids (Sudan black B reaction-SBB) and iron (Perl's reaction) was performed by a semiquantitative cytochemical method (1). A significant correlation was obtained between BUT and stage of squamous lung carcinoma (varying between I and IV) (r = 0.52, p < 0.05). There was a correlation between BUT and Perl's in healthy controls (r = 0.76, p < 0.05). The same type of correlation was observed in control smokers (r = 0.64, p < 0.05), in addition to a correlation between CHL and AcP (r = 0.69, p < 0.05). There was no significant BUT/Perl's correlation in patients with squamous cell lung carcinoma (r = 0.23, p > 0.05), but significant AcP/CHL correlation as was observed in control smokers (r = 0.73, p < 0.05), and a "new" type of correlation was shown to exist between ANAE and SBB (r = 0.77, p < 0.05). In spite of the unresolved nature of lung cancer, correlation analysis of cytochemical parameters in AM might have an important part in the analysis of their relative contribution to the development of smoking-related disorders and lung malignancies.

Establishment and characterization of a novel human cell line exhibiting both immunophenotypic markers of monocyte/macrophage and natural killer cell lineages from peripheral blood of a patient with atopic dermatitis

Large amounts of homogeneous cells are not always available for in vitro studies of inflammatory skin disorders. Here we demonstrate that a novel cell line, termed YAA, has been established from peripheral blood mononuclear cells, which were separated by the Ficoll method, of a patient with atopic dermatitis. YAA cells were grown in suspension culture. The cytochemical staining showed a positive reaction for alpha-naphthyl butyrate esterase, which was completely inhibited by sodium fluoride, but a negative result for periodic acid-Schiff, peroxidase and alkaline phosphatase. A large population of YAA cells exhibited phenotype of CD33 and CD56 but neither of CD2 nor CD3. The phorbol ester-stimulated YAA cells produced a considerable amount of tumor necrosis factor-alpha. These findings suggest that YAA might be a monocytoid line with an additional phenotype specific for natural killer cells.

Progesterone and dexamethasone stimulate proliferation and differentiation of osteoprogenitors and progenitors for adipocytes and macrophages in cell populations derived from adult rat vertebrae.

We investigated the effects of the sex hormone progesterone (Prog) and the synthetic glucocorticoid dexamethasone (Dex) on proliferation and differentiation of progenitor cells of osteogenic, adipocytic, and hemopoietic lineages in cell populations derived from explants of adult female rat lumbar vertebrae. The cell populations were obtained by culturing bone explants in plasma clots immersed in alpha-minimum essential medium plus 10% fetal calf serum (standard medium) and then subculturing the outgrowth cells in standard medium plus 50 micrograms/ml of ascorbic acid, 5 mM beta-glycerophosphate, and with or without Prog or Dex. On day 6 of culture, these populations were analyzed for cAMP responses to parathyroid hormone (PTH), prostaglandin E2 (PGE2), and isoproterenol (IPT). Increases in intracellular cAMP were seen in response to PTH, PGE2, and IPT, and culturing in medium containing Prog increased these responses. At various time periods between days 4-27 of culture, the cultures were evaluated for the presence of bone nodules, alkaline phosphatase (AP)-positive colonies, adipocytes, monocytes, and macrophages. Prog and Dex increased the number of bone nodules and AP-positive colonies. The effect of Prog on bone nodule formation was smaller than that of Dex. In addition, the effect of Dex on bone nodule formation was evident after 10 days of culture, while the Prog-induced effects became significant at days 16-20 of culture. Both hormones also increased the number of Sudan IV-positive colonies (adipocytes), certain types of alpha-naphthyl butyrate esterase (alpha-NBE)-positive colonies (monocytes, macrophages, and T-lymphocytes), and ED2-positive colonies (macrophages). Prog-treated cultures contained more colonies of small spindle-shaped alpha-NBE-positive cells and fewer colonies of small round alpha-NBE-positive cells when compared with Dex-treated cultures. These data indicate that cell populations derived from adult rat lumbar vertebrae contain, among others, osteoprogenitors and progenitors for adipocytes and macrophages that are stimulated to proliferate and differentiate by Prog and Dex. The data also suggest that the effects of Prog and Dex differ qualitatively and quantitatively.

Cytochemical staining and ultrastructural characteristics of peripheral blood leucocytes from the yellow rat snake (Elaphe obsoleta quadrivitatta)

Cytochemical staining and ultrastructural characteristics of peripheral blood leucocytes from the yellow rat snake are described. A panel of cytochemical stains, including demonstration of myeloperoxidase, acid phosphatase, naphthol AS-D chloracetate esterase, alpha-naphthol butyrate esterase and alkaline phosphatase activities; and periodic acid-Schiff and Sudan Black-B staining was performed. Snake heterophils lacked peroxidase, alkaline phosphatase and acid phosphatase activity. Azurophils stained positively for all stains except alkaline phosphatase activity. Lymphocytes showed positive acid phosphatase activity. Differentiation of thrombocytes from lymphocytes was very difficult even with cytochemical staining. Only a minor staining difference was observed with periodic acid-Schiff stain. Thrombocytes exhibited coarse, dark, purple stippling usually located in the polar area of the cytoplasm, whereas lymphocyte staining varied from none to very fine, pale pink granules dispersed throughout the cytoplasm. Ultrastructural characteristics were similar to those of mammalian leucocytes with the exception that the snake basophil granules have no crystalline matrix, and heterophil granules appeared as large, elongate, membrane bound structures of varying density with no distinct core or matrix.

1,25-dihydroxyvitamin D3 differentiates normal neutrophilic promyelocytes to monocytes/macrophages in vitro

Although it is well established that the addition of 1,25-dihydroxyvitamin D3 (D3) to the culture of normal human granulocyte/macrophage progenitors induces monocyte/macrophage (Mo/M phi) colonies, the target cells of D3 in the Mo/M phi differentiation have not been identified. We examined whether neutrophilic promyelocytes are the target cells. As a source of the promyelocyte fraction, we used colonies after 5 days of culture (5-day colonies) of colony-forming unit-granulocyte. The culture contained granulocyte colony-stimulating factor (G-CSF) as the growth factor and generated only neutrophilic colonies. The promyelocytic nature of the 5-day colonies was confirmed morphologically, cytochemically, and ultrastructurally. After morphological evaluation on part of the individual colonies, they were transferred into new semisolid cultures with or without D3 (10(-7) mol/L) in the presence of G-CSF, then incubated for the subsequent 7 days. With D3, the colonies were loose, and all the constituent cells were morphologically small macrophages, which were positive for alpha-naphthyl butyrate (alpha NB) esterase, strongly positive for CD14 antigen, and plastic-adherent. While without D3, the colonies were rather compact, and all the constituent cells were morphologically mature neutrophils, which were positive for naphthol ASD-chloroacetate esterase and weakly positive for CD14 antigen. Secondary culture of the 8- or 10-day colonies with D3 induced a lower number of alpha NB-positive cells, in proportion to the percentage of promyelocytes at the time of transfer in each colony. Four days of secondary culture with D3 was sufficient to induce alpha NB-positive cells. G-CSF was not an essential factor to induce alpha NB-positive cells. These findings indicate that D3 differentiates normal human neutrophilic promyelocytes into the Mo/M phi lineage in vitro.

Rapid screening and detection of cellular cross-contamination in cell cultures: a new application of cytochemistry.

Cellular cross-contamination is a serious problem that can potentially affect all who use cell culture systems in research and production work. Reports from national testing services indicate that frequencies as high as 17-36% of the lines examined may be incorrectly identified by source laboratories, and intra/interspecies contamination was identified in 11-25% of cell cultures examined (1). National cell banks employ a variety of techniques to attempt cell line authentication, i.e., fluorescent antibody staining, isoenzymology, cytogenetics, and, more recently, DNA fingerprinting via Southern blotting and PCR methodologies (2). Each of these procedures has advantages and disadvantages, and none is capable on a routine basis of detecting very low levels, e.g., 0.5% or less, of cross-contamination of one cell line with another from the same species. In March of 1994, a histiocyte lymphoma cell line U-937 (AKC CRL 1593) was found to be cross-contaminated with a myeloid cell line K-562 (AKC CCL243). An extraordinary, cumbersome, and expensive effort, including PCR and cytogenetics studies, was required to solve this problem. Initial stocks, after deposit, contained as little as 0.4% of the offending K-562 line (3). After proliferation for 4 weeks and many passages, the &nding cell line emerged more apparently. The U-937 line was established by C. Sundstrom and K. Nilsson in 1976 and has been extensively used to study biology of human lymphoma cells and hematological malignancies in vitro (4). In search of a rapid, reliable, and inexpensive method to screen the U-937 cell line routinely for purity and to detect early andlor minor contamination with K-562, we applied a series of cytochemical and immunocytochemical methods. We found that esterase cytochemistry is very helpful in this case and will serve in quality control for purity of large production batches. Alpha-naphthyl acetate esterase and naphthol AS-D chloroacetate esterase were present in both cell types in different amounts, as assessed by image analysis. However, alpha-naphthyl butyrate esterase was positive in U-937 and negative in K-562 cell line cells. A clear distinction on slides can be made between green-bluish (counterstain, methylene blue) K-562 cells and brown-red (esterase activity deposit) U-937 cells (Figure 1). This technique allows detection of cross-contamination with K-562 cells below 0.1%. Morphological criteria, based on Wright-Giemsa modified staining, were used in addition. The entire procedure (preparation of cells and reagents, fixation, cytochemical visualization of enzyme activity, and counterstaining) can be performed within 2 hr (5.6). We recommend the alpha-naphthyl butyrate esterase cytochemical assay as a screening procedure for detecting K-562 cell cross-contamination in U-937 cell line. This test has been applied at ATCC to a stock of U-937 now available as CRL1593.2(4). Contaminant K-562 cells have not been detected in this population. A battery of cytochemical tests can be applied on many cell lines, and specific identification characteristics can bc identified. Once such identification cards are created, cytochemistry can be used as quality control in every cell culture laboratory. This type of assessment of cell cross-contamination will save researchers from possible disappointments and will increase their confidence in the purity of cell lines. However, this application has not yet been sufficiently exploited. We hope that our letter will motivate researches to explore further applications of cytochemistry in cell culture studies.

Effect of Non-Dialyzable Extract of Amaranth Seeds on Some Cultured Leukemia Cell Lines.

The effects of a non-dialyzable extract of amaranth seeds on the biological functions of the cultured human leukemia cell lines such as monocytic U-937, promyelocytic HL-60 and macrophage-like U-M cells were examined. The extract stimulated the nitroblue tetrazolium (NBT)-reducing activity of HL-60 and U-937 cells. It was also found that the extract enhanced their α-naphthyl butyrate esterase activity. On the other hand, the NBT-reducing activity of U-M cells was not affected by the extract, but it slightly enhanced their tumor necrosis factor producing activity. These results suggest that the non-dialyzable extract of amaranth seeds contains some leukemia differentiation-inducing and macrophage-activating factors.

Acute agranular CD4-positive natural killer cell leukemia. Comprehensive clinicopathologic studies including virologic and in vitro culture with inducing agents

A 63-year-old male presented with fever, a subcutaneous nodule, gingival hypertrophy, lacrimal gland enlargement, and no lymphadenopathy or hepatosplenomegaly, but had anemia, thrombocytopenia, and peripheral blood (PB) plus bone marrow (BM) involvement by leukemic cells. There was minimal response to multiagent chemotherapy and local radiotherapy, with a survival of 6.5 months from disease diagnosis. The PB and/or BM leukemic cells were evaluated using electron microscopy (EM), immunohistochemistry, flow-cytometric immunophenotyping, cytochemistry, cytogenetics, Southern blot analysis for gene rearrangement and Epstein-Barr virus (EBV), polymerase chain reaction for EBV and human herpes virus-6 (HHV-6), and in vitro culturing with inducing agents. The leukemic cells were agranular and monocytoid, with a hairy cell-like bone marrow biopsy infiltrate. Myeloperoxidase (MPO) and alpha-naphthyl butyrate esterase staining was negative, and periodic acid-Schiff staining was positive by light microscopy. Electron microscopy showed MPO negativity and a lack of parallel tubular arrays. The immunophenotype was CD3-, CD56+, CD4+, CD8-, CD15+, TCR1-, and TCR2-, with germline immunoglobulin and T-cell receptor genes and an abnormal karyotype (44XY, 5q-, -13, 13q+, -15). No genomic material for EBV or HHV-6 was detected. Cell cultures with butyrate and N,N-hexamethylene bis-acetamide suggested the possible induction of tumor cells to express a T-cell immunophenotype. A case of clonal acute natural killer (NK) cell leukemia with an unusual morphology (agranular) and unique phenotype (CD3-, CD56+, CD4+, CD15+) is presented. Unlike as in other acute NK leukemias, EBV was negative; there was no evidence of HHV-6. The tumor cell, after culturing with differentiating agents, may have been induced to express a T-cell immunophenotype.

Effects of Glycosaminoglycans on U-937 Leukemia Cell Proliferation and Differentiation: Structure-Function Relationship

Glycosaminoglycans (heparins, dermatan sulfate, chondroitin sulfate) with different structures and physicochemical properties were evaluated for their capacity to influence proliferation and differentiation of U-937 cell line. The contrasting and specific effects of glycosaminoglycans (depending on their structures and properties) on a leukemia cell line could help explain the regulation of proliferative and/or differentiative processes of hematopoietic cells in order to clarify the control of development and differentiation of hematopoietic progenitor cells by bone marrow extracellular matrix.Heparin from beef intestinal mucosa, heparan sulfate from beef spleen, dermatan sulfate from beef intestinal mucosa, and chondroitin sulfate from bovine trachea were extracted and purified, and their purity, structures, and physicochemical properties were evaluated. Fast-moving heparin was obtained by its selective precipitation as barium salt, and partially desulfated and re-N-sulfated heparin was produced by chemical modifications. Different glycosaminoglycans were tested to evaluate their effects on proliferation and differentiation processes of a monoblastic leukemia cell line (U-937).Heparin and derivatives (from 0.1 to 100 μg/ml) inhibit cell proliferation; heparan sulfate does not produce modifications, while chondroitin sulfate and dermatan sulfate (from 0.01 to 100 μg/ml) significantly stimulate cell growth.Cell differentiation was evaluated by cytoenzymatic determination of α-naphthyl butyrate esterase and by fluorescein-labeled anti-HLA-DR, anti-CD11b, and anti-CD14 antibodies. Nitro blue tetrazolium reduction and phagocytosis were also evaluated. Heparin and derivatives significantly increase U-937 differentiation. Heparin sulfate has no effect, while chondroitin sulfate and, to a lesser extent, dermatan sulfate, induce a strong decrease of differentiative markers.The regulation of U-937 cell properties appears to be related to charge density and to the amount of N-sulfate and N-acetyl groups. In particular, glycosaminoglycans with lower sulfate-to-carboxyl ratios and N -sulfate group percentages (chondroitin sulfate and dermatan sulfate) stimulate proliferation and produce a decrease of differentiative markers; on the contrary, polysaccharides with high charge density and N-sulfate group amounts (heparin and derivatives) inhibit U-937 proliferation and induce terminal differentiation.A previous paper (N. Volpi, L. Bolognani, A. Conte, and M. Petrini, (1993) Leukemia Res . 17, 789-798) demonstrates dissimilar effects on U-937 cells by chondroitin sulfates with different structures and physicochemical properties. In this study we confirm the importance of glycosaminoglycan structures and physicochemical properties in regulating cell functions. Possible mechanisms of action are discussed.

Tuberculosis and allergic diseases

We describe here a novel unbalanced whole-arm translocation der(3;10)(q10;q10) in a 58-year-old man with acute monocytic leukemia. Bone marrow was massively infiltrated with 22.2% monoblasts, 55.4% promonocytes, and 5.6% monocytes. These monocytic cells were positive for myeloperoxidase and α-naphthyl butyrate esterase staining. Surface marker analysis revealed that they were positive for CD4, CD13, CD33, CD56, and HLA-DR but negative for CD14 and CD34. Chromosome analysis of the bone marrow cells showed 46,XY,+3,der(3;10)(q10;q10)[18]/46,XY[2]. Spectral karyotyping confirmed der(3;10)(q10;q10) as a sole structural abnormality. By acquisition of a normal chromosome 3 but not a chromosome 10, the der(3;10)(q10;q10) resulted in trisomy 3q and monosomy 10p. The +3,der(3;10)(q10;q10) is thought to play a crucial role in the pathogenesis of acute monocytic leukemia because of the gain of 3q or the loss of 10p.

Functional characteristics of in vivo induced neutrophils after differentiation therapy of acute promyelocytic leukemia with all-trans-retinoic acid

The authors describe the functional capabilities of in vivo induced neutrophils from a patient with acute promyelocytic leukemia (French-American-British M3v) treated with differentiation therapy using all-trans-retinoic acid (45 mg.m-2.day-1). The induced neutrophils from the leukemic clone appeared in the blood 7 days after therapy. Normal neutrophils, presumably derived from nonclonal normal hematopoiesis, appeared 15 days after the initiation of therapy. The induced neutrophils were separated from normal neutrophils by density gradient centrifugation. Their origin was established by fluorescence in situ hybridization. The induced neutrophils were morphologically atypical but stained for myeloperoxidase (Sudan black B) and AS-D chloroacetate esterase and were negative for alpha-naphthyl butyrate esterase. Induced neutrophils were functionally mature, showing nitroblue tetrazolium reduction in 72% of the cells compared with 84% in the normal neutrophil fraction. Both the rate and total killing of Staphylococcus aureus (American Type Culture Collection Strain 25923) were normal in both neutrophil fractions. Random locomotion was equivalent and within the normal reference range in both fractions; however, using the under-agarose technique, induced neutrophils showed a minor chemotactic defect in response to both n-formyl-methionyl-leucyl-phenylalanine (score 292, normal 338-868) and complement-derived chemotactic factors (score 420, normal 457-1408). At autopsy, induced neutrophils infiltrated necrotic myocardial tissue, suggesting a normal response to inflammatory stimuli.

ULTRASTRUCTURAL AND CYTOCHEMICAL CHARACTERISTICS OF MONOCYTES IN VARIOUS TYPES OF ACUTE LEUKEMIAS

We have examined ultrastructural and cytochemical characteristics of monocytes in lymphoblastic leukemia (ALL) patients and compared them to monocytes in acute monoblastic leukemia (AMoL) patients and acute myeloid nonmonoblastic leukemias (AML), respectively. Blood samples were prepared in the standard way for ultrastructural and cytochemical (alpha-naphthyl butyrate esterase and myeloperoxidase) analyses. Our results indicate that monocytes in ALL and acute phase AML have the same characteristics as the malignant ones in AMoL.

Establishment of a leukaemic cell line from a patient with acquisition of chromosomal abnormalities during disease progression in myelodysplastic syndrome

A cell line designated SKM-1 was newly established from leukaemic cells of a 76-year-old Japanese male patient with monoblastic leukaemia following myelodysplastic syndrome (MDS). The cells were obtained from peripheral blood of the patient when he lost multiple point mutations of ras genes with acquisition of chromosomal abnormalities during disease progression in MDS. The cells grew as a single floating cell, and have been continuously growing with the morphological characteristics of immature monoblasts by serial passages during the past 42 months with a doubling time of about 48 h. By cytochemical analysis, the cloned cells were positive for butyrate esterase, but negative for the Epstein-Barr virus associated nuclear antigen. Phenotypic analysis revealed the expression of myelomonocyte specific antigens such as CD4, CD13, CD33 and HLA-DR. Cells from the primary peripheral blood and those from 50 passages of the SKM-1 cell line both possessed no activated ras genes but showed karyotype abnormalities with 46,XY, del(9)(q13;q22), der(17) t(17;?)(p13;?). The SKM-1 cells have two mutations in p53 gene and overexpress the p53 products. This cell line may contribute to a better understanding of molecular mechanisms in the progression from MDS to myelogenous leukaemia.

Cytochemical characterization of the hemocytes of Leucophaea maderae (Dictyoptera: Blaberoidea).

A correlated morphological and cytochemical approach to the hemocytes in Leucophaea maderae characterizes the granule contents of blood cells with May Grunwald-Giemsa (MGG), periodic acid-Schiff (PAS), Alcian blue (AB), and Sudan black B stainings, and after fluorochroming with acridine orange (AO). The cell nucleus and nuclear DNA is demonstrated with the Feulgen reaction. The activities of α-naphtyl butyrate esterase (ANBE) and acid phosphatase (AcPase) have been investigated in hemocytes, by both light and electron microscopy. The study discloses five cells types: prohemocyts, plasmatocytes, granulocytes, spherulocytes, and cytocytes. Occasional Sudan black-positive cells could represent an adipohemocyte category. The different patterns of cytochemical staining suggest a variety of functional roles for hemocytes. The most selective reaction pattern in terms of cytological differentiation among hemocytes occurred with the ANBE reaction. Ultrastructural analysis of intercellular granule contents, and the demonstration of cells with intermediate features, suggests that the hemolymph of Leucophaea maderae contains a developmental series from prohemocyte to plasmatocyte to granulocyte and then to spherulocyte. © 1993 Wiley-Liss, Inc.

Classification of myeloproliferative disorders in cats using criteria proposed by the animal leukaemia study group: A retrospective study of 181 cases (1969–1992)

Bone marrow cytology in Wright or Wright Giemsa stained smears from 181 feline patients were evaluated according to recently published criteria by the animal leukaemia study group for classification of acute] myeloid leukaemas (AML) and myelodysplastic syndrome (MDS). AML was recognised in 107 (59.1%) and MDS in 39 (21.5%) cats. The frequency of acute myeloblastic leukaemia (M1 and M2 combined) was high (30.9%), erythroleukaemia (M6 and M6-Er combined) was relatively less frequent (17.1%), and acute undifferentiated leukaemia (AUL), acute monoblastic leukaemia (M5) and acute myelomonocytic leukaemia (M4) were less common, each with an incidence of less than 5.0%. Most of the M1 cats had greater than 70.0% blast cells in bone marrow compared to only a few of the M2 cats. Some blast cells in occasional AUL cats showed cytoplasmic differentiation to either erythroid or myeloid lineage or rarely to both. Type I (classical) myeloblasts predominated in all Ml cats, but occasional type II myeloblasts were seen in some cats and a few cats had some type III myeloblasts. Rubriblasts were prominent in most of the M6-Er cats. Dysplastic changes were more common in M2 than in M1 cats and included primarily dysgranulopoiesis (giant metamyelocytes and bands) and dyserythropoiesis (megaloblastic rubriblasts and rubricytes) and rarely dysmegakaryocytopoiesis (abnormal nuclear morphology). Dysplastic changes involving primarily the erythroid series were seen in M6 and M6-Er cats. Dysmyelopoiesis, in the form of occasional giant metamyelocytes and band neutrophils, was also seen in most cats considered to have myeloid hyperplasia unassociated with leukaemia or MDS. Two M2 and two MDS cats had high (>10.0%) eosinophils in bone marrow and were designated M2-Eos and MDS-Eos, respectively. Cytochemical staining was performed on bone marrow smears from 65 cases to demonstrate myeloid markers such as alkaline phosphatase (ALP), peroxidase (PO), chloroacetate esterase (CAE), and Sudan black B (SBB) and monocytic markers such as α-naphthyl acetate esterase (NAE) and a-naphthyl butyrate esterase (NBE). NAE and NBE staining reactions were usually complementary. Cytochemical heterogeneity and lineage infidelity were observed in some AML subtypes. Blast cells were positive for myeloid markers in many but not all cases of Ml and M2. Similarly monocytic markers were evident in some but not all cases of M4 and M5. Monocytic markers were also evident in blast cells of one M1 and three M2 cases, myeloid markers were present in two cases of M4, and both myeloid and monocytic markers were demonstrated in one case of AUL. Blast cells were negative for both myeloid and monocytic markers in most cases of AUL and M6. ALP-positive cells were increased distinctly (>10.0%) only in a small number of cats (15.4%) which included cases of M1, M2, M4. M6 and MDS. Prominent haematological abnormalities in most of the AML and MDS cases included moderate to severe thrombocytopenia, normocytic normochromic non-regenerative anaemia, normoblastaemia, leukocytosis accompanied by a left shift, and a variable number of circulating blast cells. Circulating blast cell counts of ⩾30.0% were seen in only 15.3% cats. Mean leukocyte counts were over 100 000/µl in most of the M5 and M4 cats and in an occasional M1 cat, whereas AUL cats generally had leukocyte counts within the normal range. Leukopenia was evident in slightly over half of the MDS-Er cats, in nearly a third of the M2 cats, and in a small number of cats in other categories. Monocytosis was most pronounced in M5 cats and prominent in M4 cats. Other abnormalities seen in a small number of cats included hyperproteinaernia or hypoproteinaemia, hyperfibrinogenaemia, elevated icterus index, and thrombocytosis.