Α-naphthyl Acetate Esterase Research Articles

SRSF2P95Hand FLT3ITDSynergistically Drives Leukemic Transformation in Zebrafish

Internal Tandem Duplication of human FMS-like tyrosine kinase (FLT3-ITD) is one of the most frequent genetic alterations in de novo acute myeloid leukemia (AML) and is often acquired during transformation of myelodysplastic syndrome (MDS) into AML, predicting an unfavorable prognosis. RNA splicing factor mutations (e.g. hot spot mutations in serine and arginine rich splicing factor 2 - SRSF2) are often found in MDS and chronic myelomonocytic leukemia (CMML). However, the clinicopathologic features of co-mutated FLT3-ITD and SRSF2 in MDS/CMML and transformation to AML are presently unclear. Here we investigated whether FLT3-ITD cooperates with SRSF2-P95H to promote leukemogenesis in zebrafish. Transgenic zebrafish carrying human FLT3-ITD or SRSF2-P95H as driven by Runx1 promoter were generated by Tol2 transgenesis. Tg(Runx1:FLT3ITD) was outcrossed with Tg(Runx1: SRSF2P95H) to generate Tg(Runx1:FLT3ITDSRSF2P95H) zebrafish. The presence of human FLT3-ITD or SRSF2-P95H was confirmed through Sanger sequencing, and the expression of each transcript was detected by qRT-PCR in both embryos and adult kidney marrow. Their clinicopathologic features were examined. At the embryonic stage, the number of leukocytes (l-plastin+) was significantly increased in Tg(Runx1: FLT3ITDSRSF2P95H) embryos compared with their single mutant and wildtype (WT) siblings, whereas the abundance of neutrophils (SBB+) was unaffected. At six months of age, Tg(Runx1:FLT3ITDSRSF2P95H) zebrafish showed monocytosis in peripheral blood (PB) (1 out of 4), but not in the single mutant and WT siblings. In kidney marrow (KM), there was expansion of both immature and mature monocytes, and dysplasia of granulopoiesis and erythropoiesis. At 9 months of age, all four Tg(Runx1: FLT3ITDSRSF2P95H) zebrafish showed neutropenia and substantial increase in monocytosis. At 1 year of age, one out of the nine Tg(Runx1: FLT3ITDSRSF2P95H) zebrafish showed a significant increase in blasts in KM, reminiscent of human AML, and the other eight Tg(Runx1: FLT3ITDSRSF2P95H) zebrafish demonstrated phenotypes consistent with human CMML-like diseases. Increased monocytic compartment of KM was confirmed by α-naphthyl acetate esterase sensitive to fluoride inhibition. In addition, both the spleen area and cellularity were expanded in Tg(Runx1: FLT3ITDSRSF2P95H) zebrafish compared with Tg(Runx1: SRSF2P95H) single mutant and WT siblings. Taken together, our findings demonstrated that FLT3-ITD cooperates with SRSF2-P95H to drive the initiation and progression of CMML and the subsequent transformation to AML. Understanding the molecular underpinnings of these events will shed light to the diverse clinicopathologic features in CMML patients.

Dual-factor Synergistically Activated ESIPT-based Probe: Differential Fluorescence Signals to Simultaneously Detect α-Naphthyl Acetate and Acid α-Naphthyl Acetate Esterase.

α-Naphthyl acetate esterase (α-NAE) and acid α-naphthyl acetate esterase (ANAE), a class of special esterases, are important for lymphocyte typing and immunocompetence-monitoring. As such, the simultaneous detection of α-NAE and ANAE has become a target to effectively improve the accuracy in lymphocyte typing. Therefore, we developed a dual-factor synergistically activated ESIPT-based probe (HBT-NA) to detect α-NAE and ANAE sensitively, rapidly, and simultaneously in a differential manner. HBT-NA exhibits differential fluorescence signal outputs toward small changes of α-NAE and ANAE activities. HBT-NA displays a weak fluorescence signal at 392 nm over a pH range from 6.0 to 7.4. However, when it interacts with α-NAE (0–25 U) at pH = 7.4, the fluorescence intensity at 392 nm enhanced linearly within 60 s (F392 nm/F0392 nm = 0.042 Cα-NAE + 1.1, R2 = 0.99). Furthermore, HBT-NA emits ratiometric fluorescence signals (F505 nm/F392 nm) for ANAE (0–25 U) at pH = 6.0 within 2.0 min, exhibiting a good linear relationship (F505 nm/F392 nm = 0.83CANAE – 1.75, R2 = 0.99). The differential fluorescence signals can be used to simultaneously detect the activities of α-NAE and ANAE in solutions and complex living organisms. More importantly, based on the differential fluorescence signals toward α-NAE and ANAE, T lymphocytes and B lymphocytes could be successfully typed and differentiated among nontyped lymphocytes, facilitating the real-time evaluation of their immune functions using flow cytometry. Hence, HBT-NA could be used for the ultrasensitive detection of the enzyme activities of α-NAE and ANAE, the real-time precise typing of lymphocytes, and the monitoring of immunocompetence.

Establishment and characterization of a DOT1L inhibitor-sensitive human acute monocytic leukemia cell line YBT-5 with a novel KMT2A-MLLT3 fusion.

Immortalized cell lines are useful for deciphering the pathogenesis of acute leukemia and developing novel therapeutic agents against this malignancy. In this study, a new human myeloid leukemia cell line YBT-5 was established. After more than 1-year cultivation from the bone marrow of a patient with acute monocytic leukemia, YBT cell line was established. Then a subclone, YBT-5, was isolated from YBT using single cell sorting. Morphological and cytogenetical characterizations of the YBT-5 cell line were determined by cytochemical staining, flow cytometry analysis, and karyotype analysis. Molecular features were identified by transcriptomic analysis and reverse transcription-polymerase chain reaction. To establish a tumor model, 5 × 106 YBT-5 cells were injected subcutaneously in nonobese diabetic/severe combined immune-deficiency (NOD/SCID) mice. DOT1L has been proposed as a potential therapeutic target for KMT2A-related leukemia; therefore, to explore the potential application of this new cell line, its sensitivity to a specific DOT1L inhibitor, EPZ004777 was measured ex vivo. The growth of YBT-5 does not depend on granulocyte-macrophage colony-stimulating factor. Cytochemical staining showed that α-naphthyl acetate esterase staining was positive and partially inhibited by sodium fluoride, while peroxidase staining was negative. Flow cytometry analysis of YBT-5 cells showed positive myeloid and monocytic markers. Karyotype analysis of YBT-5 showed 48,XY,+8,+8. The breakpoints between KMT2A exon 10 and exon 11 (KMT2A exon 10/11) and MLLT3 exon 5 and exon 6 (MLLT3 exon 5/6) were identified, which was different from all known breakpoint locations, and a novel fusion transcript KMT2A exon 10/MLLT3 exon 6 was formed. A tumor model was established successfully in NOD/SCID mice. EPZ004777 could inhibit the proliferation and induce the differentiation of YBT-5 cells. Therefore, a new acute monocytic leukemia cell line with clear biological and molecular features was established and may be used in the research and development of new agents targeting KMT2A-associated leukemia.

Auer rod-like inclusions in the cytoplasm of B-cell lymphoma cells with bone marrow infiltration

Auer rod-like inclusions are rarely seen in B-cell neoplasms patients. Here, we present a case of B-cell lymphoma with Auer rod-like inclusions in lymphoma cells. By light and transmission electron microscopy, these structures closely resembled Auer rods which were found in acute myeloid leukemia. The Auer rod-like inclusions were negative for cytochemical staining of MPO, α-naphthyl acetate esterase, and periodic acid-Schiff. Immunostaining and flow cytometric analysis confirmed monoclonal Kappa-positive malignant lymphocytes. Interestingly, the Auer rod-like inclusions exhibited diverse ultrastructural features, which ranged from fusiform, to needle-shape, to round, to crystalline cuboid, to long rod, to short rod and others; some were membrane structures, some membrane-free dense structures, and some lamellar structures. The patient was diagnosed with B-cell lymphoma with bone marrow infiltration.

Effects of stale maize on growth performance, immunity, intestinal morphology and antioxidant capacity in broilers.

ObjectiveThis study was conducted to determine the effects of stale maize on growth performance, immunity, intestinal morphology, and antioxidant capacity in broilers.MethodsA total of 800 one-day-old male Arbor Acres broilers (45.4±0.5 g) were blocked based on body weight, and then allocated randomly to 2 treatments with 20 cages per treatment and 20 broilers per cage in this 6-week experiment. Dietary treatments included a basal diet and diets with 100% of control maize replaced by stale maize.ResultsThe content of fat acidity value was higher (p<0.05) while the starch, activities of catalase and peroxidase were lower (p<0.05) than the control maize. Feeding stale maize diets reduced (p<0.05) average daily feed intake (ADFI) throughout the experiment, feed conversion ratio (FCR) during d 0 to 21 and the whole experiment as well as relative weight of liver, spleen, bursa of Fabricius and thymus (p<0.05) on d 21. Feeding stale maize diets decreased jejunum villus height (VH) and VH/crypt depth (CD) (p<0.05) on d 21 and 42 as well as ileum VH/CD on d 42. The levels of immunoglobulin G, acid α-naphthylacetate esterase positive ratios and lymphocyte proliferation on d 21 and 42 as well as lysozyme activity and avian influenza antibody H5N1 titer on d 21 decreased (p<0.05) by the stale maize. Feeding stale maize diets reduced (p<0.05) serum interferon-γ, tumor necrosis factor-α, interleukin-2 on d 21 and interleukin-6 on d 21 and 42. Broilers fed stale maize diets had lower levels of (p<0.05) total antioxidative capacity on d 42, superoxide dismutase and glutathione peroxidase on d 21 and 42, but higher (p<0.05) levels of malondialdehyde on d 21 and 42.ConclusionFeeding 100% stale maize decreased ADFI and FCR, caused adverse effects on immunity and antioxidant function and altered intestinal morphology in broilers.

Quantitative and qualitative characteristics of blood cells in black‐shouldered, Brahminy, and black kites

Black-shouldered kites (BSK, Elanus caeruleus), Brahminy kites (BrK, Haliastur indus), and black kites (BK, Milvus migrans govinda) are medium-sized hawks found in Thailand, and little is known about the hematology of these three kite species. This study reports basic hematologic values and describes the light microscopic, cytochemical, and ultrastructural characteristics of blood cells in these kites. Blood samples were collected from 113 healthy kites (50 BSKs, 53 BrKs, and 10 BKs) from January 2012 to December 2017. Complete blood cell counts, cytochemical staining (Sudan black B, peroxidase [PO], periodic acid-Schiff, α-naphthyl acetate esterase, and β-glucuronidase), and transmission electron microscopy were performed using standard methods. Hematology, morphometry, and cytochemical staining patterns of blood cells were tabulated. BSK erythrocytes were smaller than BrK and BK erythrocytes. Heterophils, the largest granulocytes, were the most prevalent leukocytes in all kites. Cytochemical reactions in blood cells from these three kite species were the same, except that heterophils from BrKs were the only cells positive for PO. The ultrastructure of heterophil and eosinophil granules from the BSKs were similar in their homogeneous electron densities but differed in shape. The eosinophil granules from BrKs and BKs revealed heterogeneous electron densities with central pallor in some granules. Basophils had different granular electron densities, and some granules were electron-lucent. The 23 baseline hematologic values and morphologic, cytochemical, and ultrastructural characteristics of all blood cell types in this study provide reference data for future kite healthcare.

Caffeic acid combined with autoclaved Leishmania major boosted the protection of infected BALB/c mice by enhancing IgG2 production, IFN-γ/TGF-β and iNO synthase/arginase1 ratios, and the death of infected phagocytes

Immunization with killed Leishmania promastigotes without adjuvant was considered as safe, but gave variable rates of protection. Taking advantage of the immuno-modulatory effect of caffeic acid (CA), a natural polyphenolic antioxidant, we investigated its potentiating effect in autoclaved Leishmania major (ALM)-induced protection of Leishmania major-infected BALB/c. First, BALB/c mice were sensitized for 6weeks either with CA, or ALM alone or combined with caffeic acid (ALM-CA) or Freund's adjuvant (ALM-FA), and subsequently infected with L. major promastigotes. Second, to test the curative effect, CA was given daily for 5weeks to susceptible mice, starting on week 4 post-infection. Sera, footpads and lymph nodes (LNs) were collected at week 9 post-infection and submitted to biochemical or histological analyses. Compared to the respective controls, our results showed that CA directly healed footpad lesions and reduced the hallmarks of cutaneous leishmaniasis including oxidative inflammation, parasite load, and phagocytes influx and infestation. In sensitized mice, the protection enhanced gradually from ALM-FA, CA, ALM to ALM-CA in parallel to decreased seric IgGt levels. In contrast to ALM-FA, the combined effect of ALM and CA increased specific isotype IgG2, and decreased IL-17 and MCP-1, and phagocyte influx, as attested by the concomitant reduction in myeloperoxidase (MPO) and α-naphthyl acetate esterase (ANAE) activities. ALM-CA shifted IFN-γ/TGF-β and iNO synthase/arginase1 (iNOS/Arg1) balances in a Th1 immune response that control efficiently cutaneous lesions and LNs hypertrophy, and reactivate the death of infected phagocytes. Therefore, CA combined with ALM synergizes with L. major antigens for priming innate cells, through early polarization to optimal Th1 response that leads to IFN-γ and iNOS-dependent leishmanicidal activity of neutrophils and macrophages.

The cytochemical and ultrastructural characteristics of phagocytes in the Pacific oyster Crassostrea gigas.

Phagocytes have been proved to play vital roles in the innate immune response. However, the cellular characteristics of phagocytes in invertebrates, especially in molluscs, remain largely unknown. In the present study, fluorescence activated cell sorting (FACS) was employed to sort the phagocytes from the non-phagocytic haemocytes of the Pacific oyster Crassostrea gigas. The cytochemical staining analysis revealed that phagocytes were positive staining for α-naphthyl acetate esterase and myeloperoxidase, while negative staining for toluidine blue and periodic acid-Schiff. The non-phagocytic haemocytes exhibited positive staining for periodic acid-Schiff, weak positive staining for toluidine blue, but negative staining for α-naphthyl acetate esterase and myeloperoxidase. In addition, phagocytes exhibited ultrastructural cellular features similar to those of macrophages, with large cell diameter, rough cell membrane and extended pseudopodia revealed by the scanning electron microscopy, while the non-phagocytic haemocytes exhibited small cell diameter, smooth cell surface and round spherical shape. Transmission electron microscopy further demonstrated that phagocytes were abundant of cytoplasmic bodies and mitochondria, while non-phagocytic haemocytes were characterized as the comparatively large cell nucleus with contorted and condensed heterochromatin adherent to the nuclear envelope. Moreover, compared with non-phagocytic haemocytes, phagocytes exhibited significantly higher levels of intracellular cytokines, including tumor necrosis factor, interferon-like protein and interleukin-17, and significantly higher abundance of lysosome and reactive oxygen species, which were of great importance to the activation of immune response and pathogen clearance. Taken together, these findings revealed the different cytochemical and ultrastructural features between phagocytes and non-phagocytic haemocytes in C.gigas, which would provide an important clue to investigate the mechanism of phagocytosis underlying the innate immune response.

Ultrastructural and Cytochemical Properties of Peripheral Blood Cells of Piebald Naked Carp (Gymnocypris eckloni).

The ultrastructural and cytochemical properties of peripheral blood cells of Gymnocypris eckloni were investigated by transmission electron microscopy and a range of cytochemical techniques to provide clear insight into the structure and function of blood cells from this fish. Ultrastructurally, erythrocytes, leucocytes (neutrophils, eosinophils, lymphocytes, monocytes), thrombocytes and plasma cells were identified in the peripheral blood of G.eckloni. The most special ultrastructural characteristics of blood cells in this fish were that neutrophils exhibited only one type of cytoplasmic granules containing an eccentric, spherical or oval electron-dense core, and eosinophils presented two types of granules with non-uniform electronic density and without crystalloids in their cytoplasm. Neutrophils, eosinophils, lymphocytes, monocytes and thrombocytes were positive for periodic acid-Schiff and α-naphthyl acetate esterase staining. Intense peroxidase positive staining was observed in neutrophils and monocytes, but not in eosinophils, lymphocytes and thrombocytes. Neutrophils, eosinophils and monocytes were stained positively for acid phosphatase, whereas lymphocytes and thrombocytes did not stain. Leucocytes and thrombocytes were negative for alkaline phosphatase and Sudan black B staining. Erythrocytes were negative for all cytochemical staining. The cytochemical and ultrastructural features of peripheral blood cells of G.eckloni were similar to those of other fish species. However, some important differences were identified in G.eckloni.

Development and characterization of a cell line from tilapia head kidney with melanomacrophage characteristics.

A novel cell line THK, derived from the tilapia head kidney, was developed and characterized. The THK cell line comprised fibroblastoid cells that markedly proliferated in Leibovitz L-15 medium containing 2%-15% fetal bovine serum (FBS) at 20°C-35°C. Cell proliferation was dependent on the FBS concentration, and the optimal temperature for proliferation ranged between 25°C and 30°C. THK cells were characterized for the presence of phagocytic activity, acid phosphatase, alkaline phosphatase, α-naphthyl acetate esterase, lipofuscin, and tyrosinase. Transcripts of CD33, CD53, CD82, CD205, macrophage colony stimulating factor receptor, GATA2, and GATA3 that are specific for leucocytes or monocytes/macrophages or both were detected in the THK cells through PCR. However, THK cells lacked for CD83, a specific marker for dendritic cells. The results indicated that the fibroblastoid THK cells were melanomacrophage-related progenitors. PCR revealed that the THK cells exhibited the transcripts of toll-like receptor 1 (TLR1), TLR2, TLR3, and CD200, of which concern with immunity as well as the transcripts of vascular endothelial growth factor receptor 3, angiomotin, and angiopoietin-like protein 2 that associate with angiogenesis regulation and macrophage proliferation. THK cells were subcultured more than 90 times and can be useful for investigating the development and functioning of the teleostean innate immune system.

Characterization of Human Breast Milk Leukocytes and Their Potential Role in Cytomegalovirus Transmission to Newborns

Background: Breast milk is the primary source of cytomegalovirus (CMV) transmission to newborns and premature infants. The role of cell-free milk whey in virus transmission is well understood, yet the knowledge about the role of milk cells in this process is scarce. Objective: To preliminarily characterize different breast milk cell types during various stages of lactation to evaluate their potential role in the transmission of CMV. Materials and Methods: Breast milk cells of 18 lactating and 3 CMV-seropositive mothers of preterm infants were isolated and characterized for expression of myeloid markers by flow cytometry. In parallel, cytospin preparations were stained with α-naphthyl acetate esterase to identify milk macrophages and describe the dynamic changes of the macrophage-granulocyte population during lactation. The influence of different time points of lactation was analyzed by FACS analysis of double-stained (CD15/CD66b) milk cells. To characterize CMV target cells in breast milk, we enriched CD14+ cells by MACS (Miltenyi) and monitored cell fractions using CMV IEEx4 nested PCR and pp67 CMV RNA by NASBA. Results: Virolactia, viral DNAlactia, and viral pp67 late mRNA could be detected in breast milk cells only in defined time periods. Granulocytes and macrophages demonstrated an inverse dynamic with neutrophils predominating in the early stages (<30 days postpartum) and macrophages in later stages (>60 days postpartum) of lactation. Enrichment of CD14-positive cells resulted in viral DNA and pp67 late mRNA detection. Conclusions: Granulocytes and monocytes/macrophages are the predominating cell populations in breast milk with changing frequencies during early lactation. These results demonstrate that CD14-positive breast milk cells seem to be one of the target cells for CMV in breast milk.

Quantitative and qualitative morphologic, cytochemical, and ultrastructural characteristics of blood cells in captive Asian water monitors.

The Asian water monitor (Varanus salvator) is the most common monitor lizard in Thailand. Reported data regarding hematology and morphology of blood cells for this species are scarce. The objective of this study was to assess routine hematologic variables and characterize the morphology, cytochemical staining, and ultrastructural features of blood cells in the Asian water monitor. Blood samples from 55 monitors (22 males and 33 females) were obtained for a CBC. Cytochemical staining (Sudan black B [SBB], peroxidase [PO], α-naphthyl acetate esterase [ANAE], and beta-glucuronidase [BG]), and scanning and transmission electron microscopy were performed using standard methods. Determined mean (range) hematologic results of all monitors included PCV 0.32L/L (0.20-0.44L/L), HGB 106g/L (62-157g/L), WBC 15.9×10(9) /L (4.0-34.0×10(9) /L), heterophil 6.3×10(9) /L (1.5-17.1×10(9) /L, azurophil 2.6×10(9) /L (0.7-9.5×10(9) /L), basophil 0.1 (0.1-0.5×10(9) /L), lymphocyte 6.8×10(9) /L (0.5-13.1×10(9) /L), and monocyte 0.2×10(9) /L (0.04-1×10(9) /L) counts. Heterophils and basophils stained strongly positive with SBB, ANAE, and BG. Heterophils contained 2 types of granules, round SBB-positive and PO-negative granules, and electron-dense, large rod-shaped granules. Gamonts of Hepatozoon sp. were found in <1% RBC of 43 monitors. There was no significant difference between hematologic variables in Hepatozoon-positive and -negative monitors. Heterophils in Asian water monitors may also function as eosinophils based on cytochemical and ultrastructural features. The quantitative results may be used as base for further studies in healthy and diseased Asian water monitors.

Optimized scorpion polypeptide LMX: a pest control protein effective against rice leaf folder.

Lepidopteran insect pests are the main class of pests causing significant damage to crop plant yields. Insecticidal scorpion peptides exhibit toxicity specific for insects. Here, we report that a peptide LMX, optimized from the insect-specific scorpion neurotoxin LqhIT2, showed high levels of activity against rice leaf folder in vitro and in planta. Oral ingestion of LMX protein led to a significant decrease in feeding on rice leaves, repression of larval growth and development, delay in molting, and increase in larval lethality. Compared with LqhIT2 protein, the stability and insecticidal efficacy of LMX was better. Meanwhile, biochemical analysis showed that LMX protein ingestion dramatically decreased ecdysone content in rice leaf folder larvae, and down-regulated enzymatic activities of the detoxification system (α-naphthyl acetate esterase and glutathione S-transferase), the digestive system (tryptase and chymotrypsin), and the antioxidant system (catalase). These changes were tightly correlated with the dosage of LMX protein. Transgene analysis showed that the rate of leaf damage, and the number of damaged tillers and leaves in the transgenic line were greatly reduced relative to wild type plants and empty vector plants. Based on these observations, we propose that the insect-specific scorpion neurotoxin peptide LMX is an attractive and effective alternative molecule for the protection of rice from rice leaf folder.

Enantioselective Interaction of Acid α-Naphthyl Acetate Esterase with Chiral Organophosphorus Insecticides

Many previous works have demonstrated that acetylcholinesterase (AChE) was enantioselectively inhibited by chiral organophosphorus insecticides (OPs) and that a significant difference in reactivation existed for AChE inactivated by (1R)- versus (1S,3S)-stereoisomers of isomalathion. It had been known that α-naphthyl acetate esterase (ANAE), an enzyme which might play an essential role in the growth of plants and the defense of plants against environmental stress by regulating the concentration of hormones in plants, can be inhibited by OPs. However, it was unknown whether interaction of ANAE with chiral OPs was enantioselective. The present work investigated the inhibition kinetics and spontaneous reactivation of ANAE inactivated by enantiomers of malaoxon, isomalathion, and methamidophos. The order of inhibition potency is (R) > (S) for malaoxon, (1R,3R) > (1R,3S) > (1S,3R) > (1S,3S) for isomalathion, and (S) > (R) for methamidophos according to bimolecular rate constants of inhibition (ki), which is consistent with the order observed in the enantioselective inhibition of AChE by malaoxon, isomalathion, and methamidophos. The difference in spontaneous reactivation of AChE inactivated between (1R)- and (1S,3S)-isomers of isomalathion is conserved for ANAE. The observations indicated ANAE and AChE have similar selective inhibition kinetics and postinhibitory reactions in reaction with chiral OPs.

Morphological and Cytochemical Studies of Peripheral Blood Cells of Schizothorax prenanti

The morphological and cytochemical studies of peripheral blood cells of Schizothorax prenanti were studied by light and electron microscopy. Erythrocytes, thrombocytes and three types of leucocytes, lymphocytes, neutrophils and monocytes, were distinguished and characterized. In addition to mature erythrocytes, immature and dividing erythrocytes were observed. A few organelles such as mitochondria were distributed in the cytoplasm of erythrocytes. Lymphocytes with heavily clumped heterochromatic nucleus and minimal cytoplasm were classified into small and large lymphocytes. Three different populations of granules, with distinctive ultrastructural aspect, were observed in neutrophils. Monocytes were the fewest leucocytes possessing rich organelles, phagocytized materials and vacuoles. Thrombocytes with various types were the most abundant blood cells among leucocytes and contained a prominent nucleus with dense bands of heterochromatin and many cytoplasmic vacuoles. Periodic acid-Schiff staining was positive in neutrophils, monocytes, lymphocytes and thrombocytes, but not in erythrocytes. Peroxidase-positive staining was observed in neutrophils and monocytes, but not in erythrocytes, lymphocytes and thrombocytes. Only neutrophils were positive for oil red O. Except for erythrocytes, the other blood cells stained positively for acid phosphatase. Only neutrophils and monocytes were positive for α-naphthyl acetate esterase. None of the cells studied were positive for alkaline phosphatase. The morphologic and cytochemical features of blood cells of S.prenanti are similar to those of other fish. This investigation may be helpful as a tool to monitor the health status of cultured S.prenanti and will grant early detection of clinical pathology.

Abstract 4959: Targeting pancreatic stellate cells (PSC) to treat pancreatic cancer.

Abstract Pancreatic cancer (PC) is characterized by a prominent stromal reaction. Recently, the contribution of the stromal elements to the initiation and progression of the disease has come to be appreciated. In this context, targeting this tumor microenvironment represents a potential strategy to improve the delivery and efficacy of chemotherapeutic agents leading a better prognosis for the patients. Pancreatic stellate cells (PSC) have been recognized as the principal cells responsible for the production of fibrosis in pancreatic cancer. In the current study, we observed that PSC possess α-naphthyl acetate esterase (ANAE) activity, which is a specific marker enzyme for monocytes and macrophages, these cells can be differentiated/activated in a similar fashion as a monocytes/macrophages, suggesting that PSC behave in a similar fashion as macrophages, and hence can be treated as such. Based on these data we targeted PSC in vitro and in vivo by using bisphosphonates (BPs) that are macrophages inhibitors. BPs inhibited proliferation and inactivated PSC in vitro through inhibition of protein prenylation, and increased cell apoptosis and cell cycle arrest in G1. Furthermore, BPs inactivated PSC, inhibited tumor growth (p&amp;lt;0.0005), tumor weight (p&amp;lt;0.001) and number of nodules (p&amp;lt;0.001) in an orthotopic animal model of pancreas. These antitumor effects were enhanced when BPs were combined with a taxane. These data suggest that BPs could disrupt the pancreatic cancer, which could offer a better prognosis to PC patients. Citation Format: Vianey Gonzalez-Villasana, Cristian Rodriguez-Aguayo, Thiru Arumugam, Zobeida Cruz-Monserrate, Defeng Deng, Enrique Fuentes-Mattei, Rosa F. Hwang, Huamin Wang, Guillermo N. Armaiz Pena, Burcu Aslan, Yolanda Gutierrez-Puente, Anil K. Sood, Craig Logsdon, Gabriel Lopez-Berestein. Targeting pancreatic stellate cells (PSC) to treat pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4959. doi:10.1158/1538-7445.AM2013-4959

Serum Chitotriosidase, a Putative Marker of Chronically Activated Macrophages, Increases With Normal Aging

Chitotriosidase (ChT) is secreted by chronically activated macrophages in Gaucher's disease. We hypothesize that circulating levels of ChT are altered with normal aging, reflecting age-related chronic macrophage activation. Potential sources that might contribute to altered levels were assessed by measuring systemic levels of ChT are α-naphthyl acetate esterase, a macrophage lysosomal enzyme; granulocyte-macrophage colony-stimulating factor (GM-CSF), which stimulates neutrophilic granule release of ChT; interleukin-6 (IL-6); and neopterin, a macrophage activation marker. Serum was obtained from 315 healthy participants whose age ranged from 18 to 92 years. Anthropometric measures included percent body fat and body mass index. ChT and α-naphthyl acetate esterase levels were measured by enzyme activity assays. GM-CSF, IL-6, and neopterin concentrations were measured by commercial enzyme-linked immunosorbent assays. Serum marker values were statistically analyzed using nonparametric tests. Six percent of the participants had undetectable ChT levels. A positive association with age was observed for ChT and IL-6, whereas a negative correlation with age was seen for α-naphthyl acetate esterase and GM-CSF. ChT values were not associated with α-naphthyl acetate esterase or GM-CSF levels. ChT was independently associated with IL-6 and neopterin levels, but statistical significance was attenuated when controlled for age. The data are consistent with increased serum ChT activity not arising from altered macrophage lysosomal enzyme trafficking or GM-CSF-stimulated release of neutrophil granule stores. The association of ChT with age remains significant after controlling for neopterin and IL-6 changes with age, suggesting that ChT levels reflect a macrophage state distinct from acute macrophage activation or inflammatory state.

Effects of vitamin E and selenium yeast on growth performance and immune function in ducks fed maize naturally contaminated with aflatoxin B1

This study was conducted to determine the effects of a combined vitamin E (VE) and selenium yeast (SeY) supplementation on growth performance, blood profiles, relative organ weight, and immune function in ducks fed diets containing maize naturally contaminated with a variety of mycotoxins, but primarily aflatoxin (AFB1). A total of 448 ducks were blocked on the basis of sex and body weight, and then randomly assigned to 1 of 4 treatments in a 2×2 factorial arrangement with 2 levels of AFB1 contaminated maize (0 or 100%) and 2 VE and SeY levels (0IU VE and 0mgSeY/kg or 50 IU VE and 0.2mgSeY/kg) for a 28-d study period. There were 7 replications (pens) per treatment and 16 birds per pen. Detectable levels of other toxins were present but that only AFB1 exceeded limits, and the level of the AFB1 was 196.8μg/kg in the contaminated maize, 130.5 and 142.4μg/kg in the 100% contaminated starter diets with or without VE and SeY, respectively, and 152.6 and 157.1μg/kg in the 100% contaminated grower diets with or without VE and SeY, respectively. Ducks fed AFB1 contaminated maize diets had lower (P<0.05) ADG and ADFI, and greater (P<0.05) death rates during the whole experiment. The supplementation of VE and SeY increased (P<0.05) FCR during d 0 to 14 but decreased (P<0.05) ADFI during d 15 to 28 and 0 to 28. Interactions were observed (P<0.05) in ducks fed AFB1 contaminated diets. Feeding AFB1 contaminated maize diets increased (P<0.05) the activity of lactate dehydrogenase, whereas the levels of total protein, albumin, and globulin decreased (P<0.05) at both d 14 and 28. However, VE and SeY had no consistent effects. The lymphocyte proliferation and acid α-naphthylacetate esterase positive ratios (ANAE+) were decreased (P<0.05) by the inclusion of AFB1 contaminated diets during the whole experiment but benefited from VE and SeY supplementation. Dietary VE and SeY improved (P<0.05) the levels of interleukin 2 (IL-2) and interleukin 6 (IL-6) throughout the experiment. Avian influenza (H5N1) antibody titer was reduced (P<0.05) by the inclusion of AFB1 contaminated maize without VE and SeY supplementation. Ducks fed AFB1 contaminated maize diets had greater (P<0.05) relative weights of spleen and bursa of fabricius but lower (P<0.05) thymus, and the supplementation of VE and SeY increased (P<0.05) the relative bursa of fabricius weight in the presence of contaminated maize. Interactive effects were observed (P<0.05) in ANAE+ and IL-2 on d 28 and relative weight of bursa of fabricius. In conclusion, feeding AFB1 contaminated maize caused adverse effects on growth performance, immunity, and relative immune organ weight in ducks, while dietary addition of VE and SeY partially counteracted the negative effects of AFB1 on growth and death rate.

Korelacija između aktivnosti alfa naftil acetat esteraze i različitog stepena suženja humane karotidne arterije

Inspite the constantly growing body of biomedical knowledge and techniques, scientists are still far from a complete understanding of molecular system dysfunctions and their relevance in atherogenesis. Accordingly, cellular non specific esterases represent a special field of research, due to their role in a wide range of diseases, including atherosclerosis. This paper deals with linear correlation between relative a-naphthyl acetate esterase (ANAE) score values and different degrees of carotid stenosis in patients with various morphological atherosclerotic lesions of this blood vessel. In our study analysis of ANAE cytochemical activity was performed in imprints of carotid plaques obtained from patients with different morphological groups of atherosclerotic lesions (n=100), as well as in control group of patients with kinking and/or coiling of carotid arteries (n=25). ANAE status was determined by cytochemical scoring system (Hayhoe and Quaglino's method modified by Cvetković, Radak and Đorđević Denić). Our results show that there is difference in relative score value of ANAE activity in carotid artery/plaque tissue between investigated groups of patients. One can, also, observed linear correlation between relative ANAE score values and different degrees of carotid stenosis in patients with various morphological atherosclerotic lesions of this blood vessel. This findings may indicate importance of ANAE in atherogenesis.

Haematological, cytochemical and ultrastructural characteristics of blood cells and serum chemistry in captive Malayan tapir (Tapirus indicus) in Thailand

The Malayan tapir (Tapirus indicus) is classified as a preserved animal of Thailand. It is listed as an endangered species on the 2008 IUCN red list and also by the Convention on International Trade in Endangered Species of Wild Fauna and Flora. Blood samples from captive Malayan tapirs (n = 20, 10 males and 10 females) from five zoos under the Zoological Park Organization of Thailand were examined and measured for complete blood counts, serum chemistry and electrolyte analysis. Cytochemical staining including Peroxidase, Sudan Black B, Periodic acid Schiff's reaction (PAS), α-naphthyl acetate esterase (ANAE) and β-glucuronidase (BG) was also analysed. Statistically significant differences were found between sexes including alkaline phosphatase, total protein and red cell distribution widths. Red blood cells were slightly anisocytosis, (range 4.5–8.5 μm) with 6.3-μm mean diameter. Eosinophils and monocytes were positive with all five cytochemical stains. Lymphocytes showed a variable reaction for ANAE and BG. PAS was positive for all type of blood cells except red blood cells. Ultrastructurally, eosinophils had the largest round shape with three-capsule layer granules which were prominent and distinguishable from other species. This investigation provides basic haematology and serum chemistry analyses as well blood cells identification guideline for the Malayan tapir and serves as endangered mammal reference values.