Α-naphthyl Butyrate Esterase Research Articles

Characterizing the Immature Immunophenotype of Sickle Cell Disease Monocytes.

Sickle cell disease (SCD) is marked by episodic vaso-occlusive crisis (VOC). Recurrent VOC creates a pro-inflammatory state that induces phenotypic alterations in innate immune cells. Monocytes are of particular interest to VOC pathophysiology because they are especially malleable to inflammatory signaling. Indeed, inflammatory disease states such as chronic obstructive pulmonary disease (COPD), obesity and atherosclerosis are known to influence monocyte development and alter monocyte subpopulations. In this study, we describe SCD monocyte subsets by performing immunophenotypic flow cytometric, enzymatic, and morphologic analysis on peripheral blood. Herein, we add to the growing body of evidence suggesting aberrant monocyte populations underpin VOC pathophysiology. We found that SCD monocytes possess an immature phenotype as demonstrated by 1) decreased CD4 positivity (p < .01), 2) low α-naphthyl butyrate esterase(ANBE) expression, and 3) naïve morphologic features. We additionally found an increase in CD14+CD16-CD4- monocytes (p < .01), a subset associated with the impaired immune response of post-trauma patients. Interestingly, we also found a large proportion of CD14+CD4-HLA-DR- monocytes which, under normal circumstances, are exclusively found in neonates (p < .01). Finally, we report an increase in nonclassical monocytes (CD14dimCD16+), a subset recently shown to have a critical role in prevention and recovery from VOC.

Characterizing the Immature Immunophenotype of Sickle Cell Disease Monocytes during Vaso-Occlusive Crisis

Sickle cell disease (SCD) is marked by episodic vaso-occlusive crisis (VOC). Painful VOC is the leading cause of emergency room visits and hospitalization in SCD, contributing heavily to the morbidity and mortality of this disease. Recurrent VOC creates a pro-inflammatory state that induces phenotypic alterations in the innate immune system. This inflammation-induced immune profile remodeling results in chronically active monocytes and neutrophils that antagonizethe vascular endothelium and further predispose SCD patients to VOC. While the mechanism of neutrophil involvement in VOC has been elucidated, less is known about the pathologic role of monocytes. Monocytes are of particular interest to VOC pathophysiology because they are especially malleable to inflammatory signaling. Indeed, inflammatory disease states such as COPD, obesity and atherosclerosis are known to influence monocyte development and alter monocyte subpopulations. Recently, nonclassical monocytes (CD14 dimCD16 +) with high heme oxygenase-1 expression have been shown to have a critical role in preventing VOC, generating renewed interest in monocytes as a therapeutic focus in SCD. In this study, we describe the monocyte subsets found in SCD patients during VOC by performing immunophenotypic flow cytometric, enzymatic, and morphologic analysis on peripheral blood obtained during hospitalization for VOC. Herein, we add to the growing body of evidence suggesting aberrant monocyte populations underpin VOC pathophysiology. We found that SCD monocytes during VOC possess an immature phenotype as demonstrated by 1) decreased CD4 positivity (p &amp;lt; .01), 2) low α-naphthyl butyrate esterase (ANBE) expression, and 3) naïve morphologic features (high nuclear/cytoplasmic ratio, indented or less lobular nuclei, and decreased cytoplasmic vacuolization). We additionally found an increase in CD14 +CD16 -CD4 - monocytes (p &amp;lt; .01), a subset associated with the impaired immune response of post-trauma patients. Interestingly, we also found a large proportion of CD14 +CD4 -HLA-DR - monocytes which, under normal circumstances, are exclusively found in neonates (p &amp;lt; .01). Finally, we report an increase in nonclassical monocytes (CD14 dimCD16 +), providing further evidence for the connection between this subset and the molecular mechanisms driving VOC. Taken together, these findings suggest that SCD patients in VOC deploy relatively immature monocytes that possess a lower capacity for antigen presentation, likely contributing to the immune dysfunction observed in SCD.

Cytochemical staining of leukocytes and platelets in the Florida manatee (Trichechus manatus latirostris): identification of a bilobed monocyte similar to other members of the Paenungulata.

Manatees (Antillean-, Amazonian, and African-) and dugongs belong to the Order Sirenia, and when combined with elephants and rock hyraxes, form the Paenungulata. A bilobed mononuclear cell has previously been identified in elephants and rock hyraxes, but not in manatees and dugongs, with cytochemical staining identifying these cells as bilobed monocytes in elephants. The objective of this study was to characterize leukocytes (white blood cells, WBC) and platelets in blood films of Florida manatees (Trichechus manatus latirostris; n = 8) using one routine hematological (Wright-Giemsa) and eight cytochemical stains: alkaline phosphatase (ALP), α-naphthyl butyrate esterase (ANBE), chloroacetate esterase (CAE), Luna, myeloperoxidase (MPx), periodic acid-Schiff (PAS), Sudan black B (SBB), and toluidine blue (TB). Heterophils and lymphocytes comprised most of the WBC, with low numbers of eosinophils, basophils, and monocytes. Additionally, 1-3% of the WBC were bilobed mononuclear cells. Bilobed mononuclear cell proportions were similar to rock hyraxes, but lower than elephants (approximate range 20-60%). Heterophils and eosinophils were positive for MPx, ALP, SBB, and PAS, with heterophils also being positive for CAE. Most of the lymphocytes were positive for ANBE and they were variably positive for CAE. Monocytes and bilobed mononuclear cells had similar cytochemical staining reactions (variably positive for all stains, except Luna and TB), supporting a monocytic origin, like elephants. Platelets were ANBE- and PAS-positive. Luna stain was useful for identifying eosinophils and TB was uninformative. This study provides new information on the morphological features and cytochemical staining characteristics of WBC and platelets and will aid in obtaining accurate hematological data of Florida manatees.

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.

Establishment and characterization of a cell line from a feline histiocytic sarcoma

Feline histiocytic sarcoma (HS) is an aggressive and uncommon tumor originating from dendritic cells/macrophages. Here, a feline HS cell line, FHS-1, was established from a case of feline HS and characterized. Immunohistochemically, FHS-1 cells were positive for vimentin and Iba-1, and negative for MHC class II and CD163. FHS-1 cells were positive for α-naphthyl butyrate esterase staining, which was clearly inhibited by sodium fluoride. FHS-1 cells had phagocytic and antigen uptake/processing activities. Moreover, FHS-1 cells were tested for susceptibility to feline infectious peritonitis virus (FIPV) strain 79-1146; however, this cell line was not susceptible to this viral strain. Although FHS-1 cells lost the expression of MHC class II and CD163, our findings indicate that FHS-1 is a feline HS cell line that retains functional properties of dendritic cells/macrophages in terms of phagocytic and antigen uptake/processing activities. While FHS-1 cells are not suitable for in vitro study of FIP using strain 79-1146, they may be applicable for studies aimed at developing new diagnostic and therapeutic strategies for feline HS.

Acute monocytic leukemia diagnosed by flow cytometry includes acute myeloid leukemias with weakly or faintly positive non-specific esterase staining.

A diagnosis of acute monocytic leukemia (AML-M5) based on α-naphthyl butyrate esterase (α-NB) staining has some problems, because AML-M5 leukemic cells often show weak or faint positivity on α-NB staining. In these situations, some cases of AML-M5 tend to be misdiagnosed as AML-M0. Therefore, we evaluated the significance of weak or faint α-NB staining in AML-M5 diagnosed by flow cytometry (FCM). Nineteen AML cases in which leukemic cells were negative for naphthol AS-D chloroacetate esterase staining were studied. For FCM, we defined leukemic cells as having a monocytic nature when more than 10% of the leukemic cells were positive for at least one of the following antigens: CD4, CD11c, CD14, and CD64. The monocytic nature determined by FCM was consistent with positive or weak positivity on α-NB staining. Five of 6 cases in which leukemic cells exhibited faint positivity for α-NB staining could be diagnosed as AML-M5 by FCM, while negative α-NB staining was consistent with a diagnosis of AML-M0. These results suggest that AML-M5 should be taken into consideration even when leukemic cells are faintly positive for α-NB staining.

Bisphosphonates inhibit stellate cell activity and enhance antitumor effects of nanoparticle albumin-bound paclitaxel in pancreatic ductal adenocarcinoma.

Pancreatic stellate cells (PSC) have been recognized as the principal cells responsible for the production of fibrosis in pancreatic ductal adenocarcinoma (PDAC). Recently, PSCs have been noted to share characteristics with cells of monocyte-macrophage lineage (MML cells). Thus, we tested whether PSCs could be targeted with the nitrogen-containing bisphosphonates (NBP; pamidronate or zoledronic acid), which are potent MML cell inhibitors. In addition, we tested NBPs treatment combination with nanoparticle albumin-bound paclitaxel (nab-paclitaxel) to enhance antitumor activity. In vitro, we observed that PSCs possess α-naphthyl butyrate esterase (ANBE) enzyme activity, a specific marker of MML cells. Moreover, NBPs inhibited PSCs proliferation, activation, release of macrophage chemoattractant protein-1 (MCP-1), and type I collagen expression. NBPs also induced PSCs apoptosis and cell-cycle arrest in the G1 phase. In vivo, NBPs inactivated PSCs; reduced fibrosis; inhibited tumor volume, tumor weight, peritoneal dissemination, angiogenesis, and cell proliferation; and increased apoptosis in an orthotopic murine model of PDAC. These in vivo antitumor effects were enhanced when NBPs were combined with nab-paclitaxel but not gemcitabine. Our study suggests that targeting PSCs and tumor cells with NBPs in combination with nab-paclitaxel may be a novel therapeutic approach to PDAC.

P3–014 - Transformed Follicular Lymphoma Mimicking Acute Lymphoblastic Leukemia

Introduction: Manifestation of the transformation of follicular lymphoma (FL) is diverse. Case: A 60-year-old man was diagnosed with FL (grade 2, clinical stage IIIA), received six cycles of R-CHOP therapy, and achieved complete remission (CR). Three years later, a follow-up CT scan revealed the regrowth of systemic lymph nodes. A clinical diagnosis of FL relapse was made and the patient received six courses of fludarabine plus rituximab, leading to a second CR. Five years later, the patient felt a mass in the right cervical region. The leukocyte count was 14,100/µl with 24% immature lymphoblasts. The bone marrow showed hyperplasia with 90.2% immature lymphoblasts. The size of blasts was small to medium, with a high N/C ratio and prominent nucleolus. Lymphoblasts were negative for peroxidase, α-naphthyl butyrate esterase, and naphthol ASD chloroacetate esterase. Immunohistochemical analysis of his bone marrow cells showed that CD10 and CD19 were positive, CD20 was slightly positive, and TdT was negative. A diagnosis of secondary acute lymphoblastic leukemia (ALL) was made based on morphologic and immunophenotypic findings. We started induction chemotherapy following the Japan Adult Leukemia Study Group (JALSG) ALL 202-O protocol. The patient's blasts showed both fusion genes IGH/BCL2 and IGH/MYC, while the primary FL sample was found to have only IGH/BCL2 fusion. Based on these findings, a diagnosis of transformed FL rather than secondary ALL was made. As the clinical course and morphology were typical of those of ALL, we continued the JALSG 202-O protocol. One course of remission induction therapy induced CR and five courses of consolidation therapy were given following the JALSG protocol, and the CR was maintained during consolidation therapy. However, the disease relapsed just after consolidation therapy was completed, and salvage chemotherapy was started. Discussion and conclusion: Transformation of FL with additional IGH/MYC gene rearrangement is well-known. However, the information on FL transformation mimicking ALL is limited. From a clinical point of view, the diagnosis of ALL should be carefully made in cases with FL.

Chinese Box turtle (Cuora flavomarginata) with lymphoid leukemia characterized by immunohistochemical and cytochemical phenotyping

Lymphoid leukemia of T-cell origin was diagnosed in a male Chinese Box turtle, Cuora flavomarginata, of approximately 25 years of age. The turtle presented with a history of anorexia, open-mouth breathing, and lethargy for one week. The CBC findings included a mildly increased PCV, and severe leukocytosis due to high numbers of atypical cells interpreted to be blasts. The blasts were medium-sized cells with round to pleomorphic nuclei, slightly clumped chromatin, indistinct nucleoli, and scant moderate-to-dark blue cytoplasm with occasional red-to-purple cytoplasmic granulation. Cytochemical and immunohistochemical staining indicated that the neoplastic cells were positive for CD3 and α-naphthyl butyrate esterase (ANBE), leading to the diagnosis of T-cell lymphoid leukemia. Histology of tissues collected at necropsy showed multifocal infiltrations of neoplastic round cells in the liver, spleen, kidneys, testicles, pancreas, thyroid, duodenum, bone marrow, epicardium, and myocardium. Transmission electron microscopy failed to identify viral particles within the neoplastic cells. This article describes the hematologic, histologic, and ultrastructural abnormalities associated with lymphoid leukemia in this turtle, and advanced diagnostic methods used for phenotyping the T-cell origin.

Human immunodeficiency virus type 1 infection alters enzymatic and ultrastructural features of peripheral blood monocytes

INTRODUCTION:Human immunodeficiency virus-1 (HIV-1) infected monocytes are now believed to serve as a reservoir for HIV-1 infection, and to play a role in viral rebound phenomena in certain groups of patients who failed or stopped highly active antiretroviral therapy (HAART). Data characterizing the morphological changes of peripheral blood monocytes in HIV-1-infected individuals are limited.MATERIALS AND METHODS:In this study, we collected monocytes from 21 asymptomatic HIV-1-infected individuals with CD4 count more than 500 cells/mm3 and healthy individuals. The monocytes ultrastructural morphologic changes and α-naphthyl butyrate esterase (ANBE) activity were compared between the two groups.RESULTS:In monocytes from patients infected with HIV-1, activity of α-naphthyl butyrate esterase (ANBE) was markedly increased compared with normal monocytes. In both light microscopic and ultrastructural studies, the cytoplasm of monocytes from HIV-1-infected patients contained a haphazard appearing network of thin fibrils. Cell surface expression of the activation marker HLA-DR molecule was upregulated. There were no discernible differences between the cell surface expression of CD4, CD14, and CD16 molecules comparing normal monocytes to those from HIV-1-infected patients. α-naphthyl butyrate esterase (ANBE) was markedly increased compared with normal monocytes. In both light microscopic and ultrastructural studies, the cytoplasm of monocytes from HIV-1-infected patients contained a haphazard appearing network of thin fibrils. Cell surface expression of the activation marker HLA-DR molecule was upregulated. There were no discernible differences between the cell surface expression of CD4, CD14, and CD16 molecules comparing normal monocytes to those from HIV-1-infected patients.CONCLUSIONS:Possibly, changes in the activity of ANBE along with a disrupted appearing cytoplasmic fibril network contribute to monocyte dysfunction in HIV-1-infected patients.

Cytochemical and immunocytochemical characterization of blood cells and immunohistochemical analysis of spleen cells from 2 species of frog, Rana (Aquarana) catesbeiana and Xenopus laevis

Mechanisms of amphibian diseases are not characterized as well as those in domestic mammalian species. Antemortem laboratory testing is limited in frogs, presenting a diagnostic challenge to zoos, laboratories, and exotic veterinarians. This study aimed to characterize blood cells and splenic cells from 2 anuran species based on characteristics identified by Wright staining, cytochemical staining, and immunochemical analysis and on histologic examination of spleens. Blood specimens and spleens were obtained from 2 species of frog, the American bullfrog (Rana [Aquarana] catesbeiana) and the African clawed frog (Xenopus laevis). Blood smears were evaluated after Wright staining and cytochemical staining for α-naphthyl butyrate esterase (NBE), chloroacetate esterase (CAE), myeloperoxidase (PER), Sudan black B (SBB), and leukocyte alkaline phosphatase (LAP) reactions and for immunoreactivity for antibodies against CD3ε, CD79a, and BLA.36 antigens. Histologic sections of spleen were evaluated after staining with H&E and for immunoreactivity for CD3ε, CD79a, and BLA.36 antigens. In bullfrogs, neutrophils, eosinophils, and monocytes were positive for some or all of the following: NBE, CAE, PER, and SBB; lymphocytes occasionally were positive for CAE. In clawed frogs, neutrophils, basophils, and monocytes were positive for some or all of the following: NBE, CAE, PER, and SBB; eosinophils occasionally were positive for CAE and PER, and lymphocytes were negative for all cytochemical stains. LAP was not a useful marker for any leukocyte type. In both species, peripheral blood lymphocytes were strongly immunoreactive for CD3ε, CD79a, and BLA.36. In splenic tissue, histologic patterns varied and there was diffuse immunoreactivity for CD79a and BLA.36 with focal reactivity for CD3ε, but with different distribution patterns in each species. Cytochemical and immunochemical analysis of cells may be helpful in identification and characterization of amphibian blood cells and splenic cells for evaluation of the health of these animals.

Cytochemistry of leukocytes from the family Macropodidae

The cytochemical staining characteristics of leukocytes from four species of Macropodidae and one species of Potoroidae were studied to investigate the cellular composition of leukocytes within the Macropodidae family and determine markers that may be useful for identifying cell lineage. Blood smears from the tammar wallaby (Macropus eugenii), the western grey kangaroo (Macropus fuliginosis), the red kangaroo (Macropus rufus), quokka (Setonix brachyurus) and a potoroid species, the woylie (Bettongia pencillata) were examined following reaction for Sudan Black B (SBB), peroxidase (PER), chloracetate esterase (CAE), α-naphthyl butyrate esterase (NBE) and alkaline phosphatase (ALP). Similar to domestic animal species, the neutrophils and eosinophils of macropodid species stained for both SBB and PER while monocytes and lymphocytes showed little or no reaction. CAE and NBE, however, were not useful as markers for macropodid neutrophils and monocytes, respectively. Significant variation between species was seen in ALP content. Tammar wallabies and quokkas demonstrated strong ALP activity within their neutrophils and eosinophils. In contrast, western grey kangaroos and red kangaroos contained no ALP activity in any of their leukocytes and staining in woylies was limited to weak reaction within some eosinophils.

A novel unbalanced whole-arm translocation der(3;10)(q10;q10) in acute monocytic leukemia

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.

Monosomy 7 as the sole abnormality of an acute basophilic leukemia

We report the case of a 72-year-old man who had the very rare disease acute basophilic leukemia with the sole chromosomal finding of a monosomy 7. Most nuclear cells in the peripheral blood and bone marrow samples were either basophils or blasts. The blasts showed negative reaction with myeloperoxidase, periodic acid Schiff, chloroacetate esterase, α-naphthyl butyrate esterase, acid phosphatase, and Sudan black B. Metachromatic features of the blasts, however, were observed with toluidine blue stain. Electron microscopic evaluation showed the typical ultrastructure, with basophil and immature mast cell granules. Cytogenetic study revealed monosomy 7 in all metaphase cells, and this finding was confirmed by fluorescence in situ hybridization. The Philadelphia chromosome was absent. Review of the literature revealed abnormalities in cases of ABL. To our knowledge, the case reported here is the first to have basophilic leukemia with monosomy 7 as the only chromosome abnormality.

Differential cytochemical staining characteristics of channel catfish leukocytes identify cell populations in lymphoid organs

This is one of the first characterizations of channel catfish ( Ictalurus punctatus) leukocytes by enzyme cytochemistry. Leukocytes demonstrated cytoplasmic staining patterns very similar to mammalian leukocytes when stained with acid phosphatase, α-naphthyl butyrate esterase, β-glucuronidase, α-naphthyl acetate esterase, Sudan Black B and anti-immunoglobulin specific immunohistochemistry. Lymphocytes, monocytes, macrophages, neutrophils, and surface immunoglobulin positive (surface Ig+) cells were present in channel catfish renal hematopoietic tissue and spleen and demonstrated distinctive cytoplasmic foci staining patterns, cytoplasmic blushing or cell membrane staining. Monocytes, macrophages, lymphocytes and surface Ig+ cells were present in the thymus. Thymic and splenic cellular organization appeared very similar to these same mammalian tissues. In the thymus, acid phosphatase positive cells were distributed throughout the parenchyma, while α-naphthyl butyrate esterase and β-glucuronidase positive cells were concentrated in the cortex and the medulla, respectively. Surface immunoglobulin positive cells occurred in the cortex. In the spleen, acid phosphatase positive cells were scattered throughout the parenchyma, while α-naphthyl butyrate esterase positive cells were scattered throughout the parenchyma and adjacent to splenic arterioles. β-glucuronidase and surface immunoglobulin positive cells were restricted to immediately adjacent to splenic arterioles. Sudan Black B positive cells were scattered throughout the parenchyma, while α-naphthyl acetate esterase positive cells occurred adjacent to peri-arteriole lymphoid sheaths and appear very similar to mammalian metallophils.

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.

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.

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.

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.