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.

Selective Butyrate Esterase Probe for the Rapid Colorimetric and Fluorogenic Identification of Moraxella catarrhalis.

Clinical identification of the pathogenic bacterium Moraxella catarrhalis in cultures relies on the detection of bacterial butyrate esterase (C4-esterase) using a coumarin-based fluorogenic substrate, 4-methylumbelliferyl butyrate. However, this classical probe may give false-positive responses because of its poor stability and lack of specificity. Here, we report a new colorimetric and fluorogenic probe design employing a meso-ester-substituted boron dipyrromethene (BODIPY) dye for the specific detection of C4-esterase activity expressed by M. catarrhalis. This new probe has resistance to nonspecific hydrolysis that is far superior to the classical probe and also selectively responds to esterase with rapid colorimetric and fluorescence signal changes and large "turn-on" ratios. The probe was successfully applied to the specific detection of M. catarrhalis with high sensitivity.

Enzyme activities as affected by mineral properties in buried volcanic soils of southern Italy

The aim of the study was to explore the likely relationships between physico-chemical and biochemical properties in buried volcanic soils of Italy, showing different degree of andic properties extent. Indeed, buried soils have the advantage to be less directly affected by anthropic impact, fresh carbon (C) inputs and environmental conditions (such as temperature, humidity, pH etc.). Two soil chronosequences, developed in different volcanic geomorphological environments, were analysed in Campania region (Italy) to assess the variations of biochemical properties according to soil andic properties. Chemical properties enabled to classify these soils as Andosols of silandic type (e.g. dominated by imogolite-type materials – ITM). As regards the relationship between soil mineral properties and biochemical activity, a positive correlation was found between enzymes and Alpy but not with Feox, Siox or ITM content. However, acid phosphomonoesterase, chitinase, leucine aminopeptidase, arylsulphatase, butyrate esterase activities and enzymes involved in C cycle (SEIc) were significantly different among soils, showing higher activity rates in soils with more pronounced andic properties. Conversely, when enzyme activity was expressed per unit of organic C (specific enzyme activity) it was significantly higher in weakly andic soils. The specific enzyme activity was particularly sensitive to the increase of Alox + 1/2Feox only for values <1%, typical of low developed andic properties, pointing to an important role of Al in controlling the interactions between the ITM mineral phase and soil enzymes. In conclusion, the soil mineralogical properties influenced the potential hydrolysis of organic C whose stabilization in the soil matrix may be affected by ITM presence and the stable interaction with soil enzymes. However, in order to establish if soils’ biochemical activity may be affected by andic properties, additional research should be addressed to cover all types of Andosols, including the aluandic ones.

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.

Morphologic and Cytochemical Characteristics of the Blood Cells of the Yellow-Bellied Slider (Trachemys scripta scripta).

The increasing prevalence of yellow-bellied sliders (Trachemys scripta scripta) as pets in the European Union and also its utilization as animal models for experimental purposes makes crucial an accurate classification of their blood cells. The aim of this work was to provide a morphologic classification based on the cytochemical characteristics of the blood cells of 15 yellow-bellied sliders. Cytochemical stains included benzidine peroxidase, chloroacetate esterase, alpha-naphthyl butyrate esterase (with and without sodium fluoride), acid phosphatase (with and without tartaric acid), Sudan black B, periodic acid-Schiff and toluidine blue. Nuclear and cellular dimensions were also measured based on quick Romanowsky-type stained smears. Besides erythrocytes and thrombocytes, five types of white blood cells were identified: heterophils, eosinophils, basophils, lymphocytes and monocytes. The cytochemical patterns of heterophils, eosinophils and basophils were unique compared to those described for other chelonians. This paper provides a useful guideline for clinical settings and further haematological studies of this species.

Sewage sludge and waterworks sludge stabilization in sludge treatment reed bed systems.

In this study, results about sludge stabilization in sludge treatment reed bed (STRB) systems in two different systems, Hanningfield STRB 1 (England), treating waterworks sludge, and Stenlille STRB 2 (Denmark), treating surplus activated sludge, are presented. The study mainly focused on the effectiveness of the STRBs systems in stabilizing sludge organic matter; in fact, parameters correlated to biochemical and chemico-structural properties of organic sludge matter were determined. Dewatering and sludge stabilization were effective in both STRBs, as highlighted by total and volatile dry solids trend. β-glucosidase, phosphatase, arylsulphatase, leucine amino-peptidase and butyrate esterase activities, enzymes related to C, P, S, N and overall microbial activity, respectively, significantly declined along the profile in both STRBs. The determination of humic carbon highlighted the formation of a stable nucleus of humified organic matter in both STRBs in the deepest layers, thus meaning the successful stabilization of sludge organic matter for both kind of sludges. Similar conclusions can be drawn from pyrolysis gas chromatography analysis (Py-GC), which enables the characterization of soil organic matter quality from a chemical-structural point of view. The pyrolytic indices of mineralization and humification showed that in both STRBs the sludge organic matter is well stabilized.

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.

Acute myeloid leukemia of mixed megakaryocytic and erythroid origin following chemotherapy for T-cell lymphoblastic lymphoma

A 14-year-old boy was admitted for fever of 3-day duration, and palpable masses in his left axilla and breast for 1 month. Four years prior to admission, he had been diagnosed with mediastinal T-lymphoblastic lymphoma without evidence of bone marrow (BM) involvement, and had received induction chemotherapy with dexamethasone, vincristine, daunorubicin, and L-asparaginase, resulting in complete remission. The patient had completed subsequent phases of consolidation and maintenance treatment and had been off treatment for 18 months before admission. On admission, PB revealed a white blood cell count of 44.07 9 10/L, a hemoglobin level of 14.3 g/dL and a platelet count of 154 9 10/L. Serum levels of lactate dehydrogenase (LDH) and uric acid were elevated to 7,490 U/L and 10.9 mg/dL, respectively. Other laboratory findings were as follows: C-reactive protein 1.78 mg/dL, AST 126 U/L, and fibrinogen [500 mg/dL. PET/CT showed increased FDG uptake in the neck, axilla, and anterior chest wall, which had not been observed at the onset of lymphoma. PB smear showed a predominance of blast cells (about 70 %). Subsequently, BM aspirate revealed 100 % cellularity with a high percentage of blasts, which accounted for 91 % of all nucleated cells (Fig. 1a, b). The medium to large-sized blasts were round to oval shaped, with finely chromatinated nuclei with or without distinct nucleoli, and scant to moderate amounts of blue cytoplasm containing some vacuoles. The blasts were dysplastic with megaloblastoid nuclei and/or bi-or multinucleated forms. They showed positivity for periodic acid-Schiff (PAS) stain in a block-like staining pattern, but were negative for myeloperoxidase (MPO) and a-naphthyl butyrate esterase (ANBE) stains (Fig. 1c). Immunohistochemistry showed that immature blasts in the BM were negative for CD61 (Fig. 1d). Immunophenotypic analysis by flow cytometry revealed that the majority of blast population was positive for CD33 and CD41a. A small portion was also positive for CD235a (glycophorin A), CD2, CD11c, CD13, and CD14. Other antigens, including CD5, CD7, CD10, CD19, CD20, CD22, CD34, CD56, CD64, CD117, cytoplasmic CD3, cytoplasmic MPO, HLA-DR, and cytoplasmic CD79a were not detectably expressed (Fig. 1e). Cytogenetic analysis of the bone marrow cells using the Giemsa banding technique revealed the karyotype 47,XY,?8,t(12;20)(p13;q13.1)[18]/46,XY[2] (Fig. 1f). FISH analysis for the RUNX1–RUNX1T1 rearrangement revealed no fusion signal, but three signals for RUNX1T1 in 90 % of the analyzed cells. Results of FISH performed using BCR-ABL1, PML-RARA, CBFB, MLL, 5q, and 7q31 probes and HemaVision (DNA Technology, Aarhus, Denmark) screening were negative. The disease was primarily classified as a therapy-related myeloid neoplasm according to the 2008 WHO classification and the secondary diagnosis of acute myeloid leukemia of mixed megakaryocytic and erythroid origin was also made based on the combination of morphology and immunophenotypic and cytochemical findings. The patient received induction chemotherapy with cytarabine and W. Jang M. Kim (&) Department of Laboratory Medicine, The Catholic University of Korea, Seoul, Korea e-mail: microkim@catholic.ac.kr

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.

Screening of microbes for novel acidic cutinases and cloning and expression of an acidic cutinase from Aspergillus niger CBS 513.88

Isolates from gardening waste compost and 38 culture collection microbes were grown on agar plates at pH 4.0 with the cutinase model substrate polycaprolactone as a carbon source. The strains showing polycaprolactone hydrolysis were cultivated in liquid at acidic pH and the cultivations were monitored by assaying the p-nitrophenyl butyrate esterase activities. Culture supernatants of four strains were analyzed for the hydrolysis of tritiated apple cutin at different pHs. Highest amounts of radioactive hydrolysis products were detected at pHs below 5. The hydrolysis of apple cutin by the culture supernatants at acidic pH was further confirmed by GC–MS analysis of the hydrolysis products. On the basis of screening, the acidic cutinase from Aspergillus niger CBS 513.88 was chosen for heterogeneous production in Pichia pastoris and for analysis of the effects of pH on activity and stability. The recombinant enzyme showed activity over a broad range of pHs with maximal activity between pH 5.0 and 6.5. Activity could be detected still at pH 3.5.

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.

Soil development and microbial functional diversity: Proposal for a methodological approach

The aim of the work was to propose a new methodological approach that relates soil microbial functional diversity to soil development under different moisture regimes. As soil evolution proceeds through an increasing niche separation we expect a link between functional diversity and soil development. Shannon's (H') and Gini (D) diversity indices were calculated using eight enzyme activities (β-cellobiohydrolase, N-acetyl-β-glucosaminidase, β-glucosidase, α-glucosidase, acid phosphatase, arylsulfatase, xylosidase and butyrate esterase) in order to assess functional diversity at different scales, from soil horizons (α-diversity) to soil profiles (β-diversity) under different moisture regime (γ-diversity) and belonging to different taxonomic levels. In addition, the ratio of acid phosphatase to chitinase was calculated as a potential index of soil development. Eight soil profiles were selected: four in Northwestern Italian Alps or Northern Apennines with Udic soil moisture regimes (Typic Haplocryod, Mollic Haplocryept, Lithic Dystrudept, Lithic Cryorthent) and four in Northeastern Italy where in two cases the water table near the soil surface strongly affects the Ustic soil moisture regime, and intrazonal Aquic regime or Aquic conditions develop (Typic Haplustept, Typic Ustipsamment, Aquic Ustipsamment, Typic Psammaquent). D ranged from 50 to 95%, while the H' ranged from 3 to 2.4 in Lithic Cryorthent and Typic Psammaquent, respectively. Under Udic moisture regime an inverse relationship between soil profile development and the diversity index was observed. However the lowest the diversity in the profile, the highest the variability of the values obtained within horizons in the soil profile suggesting a link between differentiation of soil horizons and biochemical properties. The Aquic conditions interfere in establishing the relationship between soil profile development and the microbial functional diversity since H' and D increased in Typic Psammaquent with respect to Typic Haplustept (H' from 2.4 to 2.6 and D from 52.2 to 60.4). Finally, the phosphatase/chitinase ratio was related to soil development since the lowest values were obtained in the upper horizons of Typic Haplustept, Typic and Aquic Ustipsamment (from 2.0 to 4.0), while the highest values were obtained in deep horizons of Typic Haplocryod and Lithic Dystrudept (e.g. 39.2 in Bs2 and 28.0 in Bw1). In conclusion, microbial functional diversity assessed using Shannon or Gini diversity indices and phosphatase/chitinase ratio measured at different scales from soil horizons to soil profiles under different moisture regime and belonging to different taxonomic levels, may represent a new approach to establish the interrelationship between pedogenetic processes, soil development and soil microbial functions.

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.

Stratification of selected hydrolytic enzyme activities in the sediment in two lakes in Finland

To investigate the stratification of hydrolytic enzyme activities and importance of subsurface layers in depolymerization of detritus biopolymers, nine hydrolytic enzyme activities involved in the cycling of carbon, phosphorus, nitrogen and sulphur were measured in various sediment layers and their extracts at Kyläniemi in Lake Saimaa in southern Finland and in Lake Ahvenjärvi in northern Finland. The results show that for each lake all nine hydrolytic enzyme activities were higher in all sediment layers than in comparable sediment extracts indicating that the major part of enzymes was bound to the sediment particles in all layers in both lakes. Carbohydratase, β-cellobiosidase, activities did not show any gradient with sediment depth at Kyläniemi in Lake Saimaa indicating that there was rapid turnover of carbohydrates in the entire sediment column. The activities of acetate esterase, butyrate esterase, phosphomonoesterase, aminopeptidase, N-acetyl glucosaminidase, sulphatase and β-glucosidase in the deepest layers were 19–53% of those in the surface sediment indicating that depolymerization of biopolymers involved in the cycling of carbon, nitrogen, phosphorus and sulphur was substantial in subsurface sediment.

Focal Positivity for Non-Specific Esterase in AML: A Surrogate Marker to Predict AML with t(8;21)/RUNX1-RUNX1T1

Abstract 2534Introduction Cytomorphological evaluation of bone marrow remains a fast and easy accomplished tool in patients with suspected acute myeloid leukemia (AML). Some cytogenetic aberrations can be predicted by specific morphological features like abnormal eosinophils associated with inv(16)/t(16;16) and faggot cells with t(15;17). But implementations of cytogenetic and molecular tests nearly diminished the interest in studying cytomorphological features that might be associated with chromosomal aberrations. Common morphological characteristics often found in AML associated with t(8;21) are large blasts with abundant basophilic cytoplasm, containing numerous azurophilic granules, perinuclear clearing or hofs and single long and sharp Auer rods. But not all AML with t(8;21) show these characteristic signs. During our cytomorphological review for patients with AML within the German prospective multicenter randomized AML96 and AML2003 trials we observed that a focal non-specific esterase stain for blasts in t(8;21) appears to be another specific feature for this subentity. With this study we tried to analyze its predictive value for this chromosomal aberration. Materials and methodsBone marrow (BM) aspirates and peripheral blood smears of 156 eligible patients from the Study Alliance Leukemia (SAL) AML96 and AML2003 trials were investigated by conventional cytochemical methods (May-Giemsa, myeoloperoxidase, alpha-naphthyl butyrate esterase and iron staining). AML classification was performed according to FAB and WHO 2001. Diagnosis of t(8;21)/RUNX1-RUNX1T1 was made by FISH, chromosomal banding and RT-PCR analysis. Bone marrow samples from patients with AML associated with t(8;21)/RUNX1-RUNX1T1 were evaluated and reviewed by an independent and experienced morphologist. Blast morphology was evaluated for granulation, perinuclear clearing or hofs, focal positivity for non-specific esterase (NSE) and the presence of Auer rods, pseudo-Pelger neutrophils and eosinophils. Besides that we investigated its correlation for aberrant expression of CD19 and CD56 in immunophenotyping and the KIT exon-17 mutation. All statistical calculations were performed using the software program PASW statistics 17 (SPSS, Chicago, IL, USA) and R 2.10.1 (The R Foundation for Statistical Computing, 2010). ResultsOf 156 eligible patients, 72 did not have adequate bone marrow samples to assess the amount of blasts with NSE positivity; therefore, the final study cohort consisted of data sets from 84 patients. The median age was 44 years (range, 19–85 years). The distribution of gender was 39 women (46%) and 45 men (54%). The majority of cases (81%) were subclassified in FAB M2. Cytomorphology showed a focal NSE positivity in 82 patients (98% of cases). Chromosome banding analyses were performed in all cases and showed t(8;21) in all cases. Forty-three patients demonstrated additional abnormalities with 29 (35%) cases showing single additional aberrations and 14 (17%) cases having two or more additional aberrations. Focal positivity of non-specific-esterase stain was found in 25% in 11 (13%), 50% in 29 (35%), 75% in 22 (26%) and 100% in 20 (24%) of bone marrow blasts. The presence of focal NSE-positivity demonstrates a significant association with the cytogenetic aberration t(8;21) (p < 0.001). Focal NSE-positivity was present even in the absence of further morphological features associated with t(8;21). No correlation between focal NSE stain and the aberrant expression CD19 and CD56 and KIT-Mutation could be seen. In AML other than t(8;21) no focal positivity for NSE could be seen (n=100). ConclusionOur investigations could prove that focal positivity for NSE is a reliable surrogate marker to predict the chromosomal aberration t(8;21). Disclosures:No relevant conflicts of interest to declare.