AS-D Chloroacetate Esterase Research Articles

Diurnal changes in bacterial settlement on the Peyer's patch and surrounding mucosa in the rat ileum and its effect against the intestinal immune system.

Our previous study revealed the diurnal change in the indigenous bacteria settling on the terminal region of the rat ileum. In the present study, we investigated the diurnal change in indigenous bacteria on the most distal ileal Peyer's patch (PP) and surrounding ileal mucosa and explored how stimulation from indigenous bacteria for a day affects the intestinal immune system at the beginning of the light phase. Histological measurement revealed that bacteria adjacent to the follicle-associated epithelium of PP and to the villous epithelium of the surrounding ileal mucosa are more abundant at zeitgeber time (ZT)0 and ZT18 than at ZT12. On the other hand, tissue-section 16S rRNA amplicon sequencing revealed no significant difference between ZT0 and ZT12 in the bacterial composition on the ileal tissue including the PP. One-day treatment with an antibiotic (Abx) successfully impaired the settlement of bacteria around the ileal PP. In transcriptome analysis, 1-day Abx treatment led to the downregulation of several chemokines in both PP and ordinary ileal mucosa at ZT0. Histological analysis of the 1-day Abx group revealed decreases in both CD68+ macrophages in PP and naphthol AS-D chloroacetate esterase stain-positive mast cells in the ileal villi. Together, these findings suggest that the colonies of indigenous bacteria on the distal ileal PP and surrounding mucosa expand during the dark phase, which might lead to the expression of genes to regulate the intestinal immune system and contribute to the homeostasis of at least macrophages in PP and mast cells in the ileal mucosa.

A rare der(10)t(X;10)(p11.2;p11.2) in an elderly patient with therapy-related acute myelomonocytic leukemia.

An 80-year-old male was referred to our hospital with dyspnea on effort. He had a history of diffuse large B-cell lymphoma 2 years ago, for which he was treated with chemotherapy, including rituximab, cyclophosphamide, vincristine, doxorubicin, and prednisolone. His laboratory data included a white blood cell count of 1.9 × 109/L, a hemoglobin concentration of 10.5 g/dl, and a platelet count of 92 × 109/L. His serum lactate dehydrogenase level was 196 U/L. A bone marrow examination revealed a hypercellular bone marrow with a blast frequency of 23.4%. The blast cells were positive for both nonspecific esterase and naphthol AS-D chloroacetate esterase staining (Figure 1). A cytogenetic study of the patient's bone marrow cells demonstrated the following karyotype: 47,XY, +8, der(10)t(X;10)(p11.2;p11.2)[7]/46,XY[13] (Figure 2). A diagnosis of therapy-related acute myelomonocytic leukemia was made due to his history of chemotherapy-treated lymphoma. Although the patient received two courses of azacitidine monotherapy, it was not effective, and he died 3 months after being diagnosed with leukemia. To the best of our knowledge, only five cases of acute myeloid leukemia (AML) involving a translocation consisting of 10p11 and Xp11 breakpoints have been reported [1-5]. Of these, one case demonstrated the t(X;10)(p11;p11) translocation as a sole abnormality [3]. This translocation has never been reported in other solid tumors. Hence, t(X;10)(p11;p11) is considered to be a rare, but recurrent, cytogenetic aberration in AML. The reported cases have several things in common, that is, they all involved balanced translocations, most of them involved therapy-related leukemia, and all of the patients except one (a 50-year-old [5]) were children. Although our case also involved therapy-related leukemia, it was very rare, as it involved an 80-year-old patient who harbored an unbalanced der(10)t(X;10)(p11;p11) translocation. As for the association between t(X;10)(p11;p11) and the pathogenesis of AML, our case involved der(10)t(X;10)(p11;p11), but lacked der(X)t(X;10)(p11;p11) due to the unbalanced nature of the translocation. Therefore, der(10)t(X;10)(p11;p11), rather than der(X)t(X;10)(p11;p11), is proposed to play a crucial role in the leukemogenesis in t(X;10)(p11;p11)-harboring AML. The patient's consent was obtained for the publication of the case report. The authors declare no conflict of interest. Masahiro Manabe, Nao Tanizawa, Satoru Nannno, and Ki-Ryang Koh designed the study. Masahiro Manabe, Yuuji Hagiwara, and Reiko Asada analyzed the data. Masahiro Manabe and Ki-Ryang Koh collected the clinical data and specimens. Masahiro Manabe, Yuuji Hagiwara, and Reiko Asada wrote the manuscript. All of the authors have reviewed and approved the final manuscript. All data generated during this study are included in this manuscript.

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.

Diagnosis and treatment of adult acute myeloid leukemia with NPM-MLF1 fusion gene and literatures review

Objective To evaluate the clinical features, treatment and prognosis of acute myeloid leukemia (AML) with nucleophosmin (NPM)-myelodysplasia/myeloid leukemia factor (MLF)1 fusion gene. Methods On 16 February 2016, a case of AML patient with NPM-MLF1 fusion gene who was admitted to Changhai Hospital Affiliated to Second Military Medical University was included in this study. When the patient was admitted, bone marrow morphology, cytochemical staining and immunophenotype analysis of leukemia blasts, cytogenetics and molecular genetic analysis were conducted and diagnosis was made base on those test results. On 17 February 2016, the patient recieved two courses of daunorubicin+ arabinosylcytosin (DA) 3+ 7 induction therapy. Subsequently, the patient recieved one course of fludarabine, cytarabine and granulocyte colony-stimulating factor (FLAG) regimen and 5 courses of high-dose of cytarabine. In the meantime, bone marrow morphology and minimal residual disease (MRD) were conducted during the chemotherapy. On 6 April 2017, the patient received bone marrow morphology, immunophenotype analysis of leukemia blasts and molecular genetic analysis on her visit. The clinical features, diagnosis and treatment of this patient were analyzed retrospectively, and the related literatures were reviewed. Results ①When the patient was admitted, bone marrow morphology revealed hyper-cellular marrow with 48.0% leukemia blasts. Cytochemical staining showed that peroxidase (POX) staining in some leukemia blasts were positive, and naphthol AS-D chloroacetate esterase (CE) staining in a few leukemia blasts were positive, and non-specific estertase (NSE) staining in some leukemia blasts were positive. The results of immunophenotype analysis revealed the ratio of the blasts cell was about 39.9%, and the ratio of immature cell was about 13.6%. R banding karyotype analysis marked with 46, XX, t(3; 5)(q25; q34)[3]/46, XX[7]. Molecular genetic analysis showed NPM/MLF1 fusion gene was positive. According to above, the diagnosis of patient was AML-subtype M4 with NPM-MLF1 fusion gene. ②After the patient was treated with the first course of induction therapy, and the reexamined bone marrow morphology post therapy showed partial remission (PR). The patient received complete remission (CR) after the second course of induction therapy. After the patient received consolidation therapy, and bone marrow morphology showed the patient obtained sustained CR status. On the patient′s last visit, bone marrow morphology revealed a total of 25.0% leukemia blasts, consisting of 21.0% blasts and 4.0% immature cell, suggesting AML recurrenced. Immunophenotyping of leukemia blasts showed approximately 28.5% of the early myeloid cells in bone marrow which was consistent with AML recurrence. Molecular genetic analysis showed that the MPM-MLF1 fusion gene was positive (66.1%). During the treatment, bone marrow MRD decreased, then increased. The patient went back to local hospital for treatment after AML relapsed. Conclusions AML-subtype M4 patient with NPM-MLF1 fusion gene can achieve CR after chemotherapy, but prone to early recurrence, and NPM-MLF1 fusion gene may be a poor prognostic factor for AML. Key words: Leukemia, myeloid, acute; Drug therapy; NPM-MLF1; t (3;5)

ACTIVITY OF LEUKOCYTE ENZYMES IN AMERICAN MINK (NEOVISON VISON): GENOTYPIC AND AGE-RELATED DIFFERENCES

The activity and localization of enzymes in blood leukocytes were examined in dark brown (+/+), silver-blue (p/p) and sapphire (a/a p/p) minks during several periods of postnatal development. This study was performed by morphometric and cytochemical methods using light microscopy and image analysis software “VidеoTest». Significant genotypic differences were determined in the distribution of peroxidase, naphthol AS-D chloroacetate esterase (NASDCE) and alkaline phosphatase (AP) in leukocytes. Sapphire mink (a/a) are characterized by the lowest reproductive potential and increased postnatal mortality among the studied genotypes. Leukocytes of sapphire mink showed a significant increase in the size of and a change in the number of peroxidase- and esterase-positive structures, as well as the presence of phosphatase-negative areas in the cytoplasm. Apparently, these structures are abnormal granules and indicate the disturbance of granulogenesis. Mink of all the three genotypes demonstrated similar age-related changes of peroxidase and alkaline phosphatase activities. The lowest peroxidase activity was observed in early postnatal development, whereas the highest – at the age of 120 days. The increase in alkaline phosphatase activity occurred during the phase of active growth – on the 60th day of postnatal ontogeny. The pattern of age-related changes in NASDCE activity depended on the mink’s genotype. Sapphire mink had the highest level of NASDCE activity at the age of 4 days and the lowest – at 180 days, whereas both dark brown and silver-blue mink at the same age (180 days) had the highest values of NASDCE activity. The postnatal dynamics of the enzymes’ activity in the three genotypes apparently reflects the specific features of metabolism in leukocytes in different periods. The cytochemical features of peripheral blood leukocytes in sapphire mink suggest the presence of a morphofunctional cellular defect, which is the cause of the animals’ low viability.

Clinicopathological features of myeloid sarcoma: Report of 39 cases and literature review

ObjectiveTo explore the clinicopathological features of myeloid sarcoma (MS). MethodsWe retrospectively analyzed the clinicopathological features of patients with MS and reviewed the relevant literature. ResultsThere were 39 patients (20 male and 19 female, with a ratio of 1.1:1) aged 2–62 years (median: 33 years; mean: 33.4 years), with 53.9% patients in the 21–50 years age group. The clinical manifestations varied and were dependent on the lesion location. Immunohistochemistry and special staining showed that the positive rates for myeloperoxidase, CD43, CD34, CD117, CD68, Lysozyme, CD99, and naphthol AS-D chloroacetate esterase were 92.1% (35/38), 91.3% (21/23), 44.8% (13/29), 42.3% (11/26), 61.1% (11/18), 100.0% (5/5), 78.6% (11/14), and 55.6% (10/18), respectively. Histologically, the cells were predominantly arranged diffusely, and the solitary infiltrating cells may show an acinus-like arrangement, while some cells were arranged in linear or “Indian file” pattern. Obvious small nucleoli were seen in most cases. The nuclei were predominantly round or oval, although eccentric, kidney-shaped, and lobulated nuclei were also observed. A varied number of eosinophilic granulocytes were found in most cases. The “starry sky” appearance was observed in a few patients. Among 18 patients, the survival rate was 38.9% and the median survival time was 28 months. MS occurred following allogeneic stem cell transplantation in 5 cases. There were 14 de-novo (45.2%,14/31) and 17 secondary MS (54.8%, 17/31). As for de-novo MS, female rates was higher (64.3% vs. 29.4%), older average years (41.4 vs. 31.1 years), older middle years (47.5 vs.32 years). ConclusionsThe combined application of morphology, immunohistochemistry, and special staining may facilitate the diagnosis of this malignancy. Surgery plus chemotherapy remains the most common treatment for MS. The prognosis of MS was bad.

Morphology and staining behavior of neutrophilic and eosinophilic granulocytes of the common marmoset (Callithrix jacchus)

Common marmosets (Callithrix jacchus) are frequently used as translational animal models for human diseases. However, a comparative study of cytological and histochemical detection methods as well as morphometric and ultrastructural characterization of neutrophils and eosinophils in this species is lacking. Blood samples of house dust mite sensitized and allergen challenged as well as lipopolysaccharide (LPS) challenged marmosets were analyzed with different cytological and histological staining methods. Furthermore, cell size and number of nuclear segments were compared between neutrophils and eosinophils. Electron microscopy was performed to characterize the ultrastructure of granulocytes. Of all applied cytological stains, three allowed differentiation of eosinophils and neutrophils and, thus, reliable quantification in blood smears: May-Grünwald-Giemsa stain, Congo Red and Naphthol AS-D Chloroacetate-Esterase. For histology, Hematoxylin-Eosin (H&E) could not demonstrate clear differences, whereas Sirius Red, Congo Red, and Naphthol AS-D Chloroacetate Esterase showed capable results for identification of eosinophils or neutrophils in lung tissue. Morphometry revealed that marmoset neutrophils have more nuclear segments and are slightly larger than eosinophils. Ultrastructurally, eosinophils presented with large homogeneous electron-dense granules without crystalloid cores, while neutrophils were characterized by heterogeneous granules of different size and density. Additionally, sombrero-like vesicles were detected in tissue eosinophils of atopic marmosets, indicative for hypersensitivity-related piecemeal degranulation. In conclusion, we provide a detailed overview of marmoset eosinophils and neutrophils, important for phenotypic characterization of marmoset models for human airway diseases.

Abstract 720: Edaravone Reduces Kidney Damage Induced by Myonephropathic Metabolic Syndrome in Rats

Background: Free radicals cause myonephropathic metabolic syndrome (MNMS), which damages not only muscles but also kidneys. At ATVB 2014, we previously reported edaravone (Radicut®, Mitsubishi Tanabe Pharma Co., Japan), the free radical scavenger may suppress kidney damage induced by MNMS. Objective: In this study, we evaluated whether edaravone suppresses MNMS-induced kidney damage, focusing on active neutrophils and tubular function. Methods: Either 3.0 mg/kg of edaravone (edaravone group; n = 4), or saline (MNMS group; n = 6) was intraperitoneally injected into male Lewis male rats (508 ± 31 g). MNMS models were induced, by clamping the bilateral common femoral arteries for 5 hours, followed by de-clamping after more 5 hours. The both kidneys were stained with naphthol AS-D chloroacetate esterase to detect active neutrophils and with Periodic Acid Schiff (PAS) to assess glycogen storage. Normal kidneys were also stained as control (n = 3). Kidney damage was evaluated by counting the number of chloroacetate esterase-positive cells (neutrophil infiltration) and the percentage of PAS-positive area (tubular function). Results: The number of chloroacetate esterase-positive cells was significantly increased in the MNMS group than that in normal kidneys (62.4 ± 5.6 vs. 17.5 ± 2.4 cells/mm2, p<0.01). However, the number of chloroacetate esterase-positive cells was significantly reduced in the edaravone group (32.8 ± 5.7 cells/mm2, p<0.01, Figure attached). The percentage of PAS-positive area in the MNMS group was significantly decreased than that in normal kidneys (36.9 ± 2.6 vs. 66.9 ± 1.2%, p<0.01). The percentage of PAS-positive area was maintained in the edaravone group (52.9 ± 0.9 %, p<0.01, Figure attached). Conclusions: Edaravone may reduce neutrophil infiltration around the glomerulus and tubular dysfunction induced by MNMS.

Edaravone injected at the start of reperfusion suppresses myonephropathic metabolic syndrome in rats.

The purpose of this study was to evaluate whether edaravone (Radicut(®), Mitsubishi Tanabe Pharma Co., Osaka, Japan) injected at the start of reperfusion can suppress myonephropathic-metabolic syndrome (MNMS). MNMS models were made by clamping the bilateral common femoral arteries for 5 hours. At de-clamping (at the start of reperfusion), they were intra-peritoneal injected with 9.0 mg/kg of edaravone (the edaravone group, n = 5) or an equal volume of saline (the control group, n = 5). At five hours after de-clamping, the lower extremity muscles were stained with hematoxylin & eosin (H&E) to count the viable cells, and periodic acid- Schiff (PAS) to assess the glycogen storage. The lungs were also stained with H&E to expresse the alveolar wall thickness, and naphthol AS-D chloroacetate esterase to label infiltrating active neutrophils. The viable muscle cells in the edaravone group was significantly greater than that of the control group (593 ± 60 vs. 258 ± 31 cells/mm(2), p < 0.01). The PAS-positive area in the edaravone group was also significantly higher than that in the control group (30.1 ± 6.9 vs. 7.3 ± 2.1%, p < 0.001). The alveolar wall thickness in the edaravone group was significantly lower than that in the control group (63.6 ± 5.6 vs. 17.2 ± 5.2%, p < 0.001). The active neutrophil infiltration in the edaravone group was also significantly lower than that in the control group (249 ± 59 vs. 68 ± 8 cells/mm(2), p < 0.001). We conclude that edaravone injected at the start of reperfusion can suppress not only muscle reperfusion injury but also lung damage.

Ethyl pyruvate decreases airway neutrophil infiltration partly through a high mobility group box 1-dependent mechanism in a chemical-induced murine asthma model.

Diisocyanates are one of the leading causes of occupational asthma, which is dominated by granulocytic inflammation in the airway. In this study, we intended to explore the role of ethyl pyruvate (EP) on neutrophil infiltration in a toluene-2,4-diisocyanate (TDI)-induced murine asthma model. The experimental mice were first dermally sensitized and then challenged with TDI via oropharyngeal aspiration. The mice were treated intraperitoneally with 100, 50 or 10mg/kg EP 1h before each challenge. One day after the last challenge, airway reactivity to methacholine was measured by a barometric plethysmographic chamber. Total and differential cell counts, along with levels of macrophage inflammatory protein-2 (MIP-2), TNF-α in bronchoalveolar lavage (BAL) fluid and mRNA expression of CXCR2 in the lung were assessed. To depict neutrophils, a naphthol AS-D chloroacetate esterase kit was used. High mobility group box 1 (HMGB1) was determined by western blot and immunohistochemistry. Treatment with EP dramatically decreased airway hyperresponsiveness in TDI-challenged mice, as well as numbers of neutrophils in BAL fluid and peribronchovascular regions. Both the TDI-induced raised protein level and abnormal distribution of HMGB1 were significantly recovered by EP in a dose-dependent manner. The concentration of MIP-2 in TDI-induced asthma mice was significantly higher than that of the control ones, while EP had few effects on MIP-2. The mRNA expression of CXCR2 didn't change significantly, and TNF-α was not detected in BAL fluids. EP reduces airway neutrophil infiltration partly through downregulating HMGB1 in a chemical-induced murine asthma model.

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.

Intravenous Immunoglobulin-Induced Neutrophil Apoptosis in the Lung during Murine Endotoxemia

The pathophysiologic features of acute respiratory distress syndrome (ARDS) are attributed to neutrophil accumulation and over-activation. Low blood immunoglobulin G concentrations in septic shock patients are associated with higher risk of developing ARDS. This study showed the effects of intravenous immunoglobulin (IVIg) on neutrophil apoptosis and accumulation in the lung during murine endotoxemia. Male C57BL/6J mice were injected with saline or 7 mg/kg of lipopolysaccharide (LPS), and 3 h later also were injected with saline, IVIg 300 mg/kg, or IVIg 1000 mg/kg intraperitoneally. At 12 h after LPS injection, mice were sacrificed and peripheral blood and lungs were collected. The lung messenger ribonucleic acid expression (tumor necrosis factor-α [TNF-α], inducible nitric oxide synthase [iNOS], and intercellular adhesion molecule-1 [ICAM-1]) was determined using quantitative realtime reverse transcriptase-polymerase chain reaction. Lungs were immersed in 4% paraformaldehyde and then embedded in paraffin. Tissue slices were prepared and stained with naphthol AS-D chloroacetate esterase to detect neutrophils. The numbers of neutrophils (characterized by the segment number of their nuclei) were counted. Peripheral neutrophil apoptosis was detected by annexin V using flow cytometry and lung neutrophil apoptosis was detected by cleaved caspase-3 using immunohistochemistry. The survival rates of the saline group, LPS group, and IVIg group were all 100%. Apoptosis of peripheral blood neutrophils was inhibited by LPS. Neutrophil accumulation in the lung was decreased by both IVIg 300 mg/kg and 1000 mg/kg. Segmented neutrophils were reduced by IVIg during endotoxemia. However, IVIg 300 mg/kg and 1000 mg/kg had no influence on the lung messenger ribonucleic acid expression of TNF-α, iNOS, or ICAM-1. Cleaved-caspase-3-positive neutrophils were increased in the IVIg 300 mg/kg group during endotoxemia. The 1000 mg/kg IVIG dose reduced the number of segmented neutrophils, but did not induce cleaved-caspase 3-positive neutrophils. A therapeutic IVIg dose can attenuate neutrophil accumulation and regulate neutrophil apoptosis in the lung during endotoxemia. It is possible that the pathways by which IVIG induces neutrophil apoptosis may differ depending on the IVIg concentration.

Lysophosphatidic acid triggers mast cell-driven atherosclerotic plaque destabilization by increasing vascular inflammation

Lysophosphatidic acid (LPA), a bioactive lysophospholipid, accumulates in the atherosclerotic plaque. It has the capacity to activate mast cells, which potentially exacerbates plaque progression. In this study, we thus aimed to investigate whether LPA contributes to plaque destabilization by modulating mast cell function. We here show by an imaging mass spectrometry approach that several LPA species are present in atherosclerotic plaques. Subsequently, we demonstrate that LPA is a potent mast cell activator which, unlike other triggers, favors release of tryptase. Local perivascular administration of LPA to an atherosclerotic carotid artery segment increases the activation status of perivascular mast cells and promotes intraplaque hemorrhage and macrophage recruitment without impacting plaque cell apoptosis. The mast cell stabilizer cromolyn could prevent intraplaque hemorrhage elicited by LPA-mediated mast cell activation. Finally, the involvement of mast cells in these events was further emphasized by the lack of effect of perivascular LPA administration in mast cell deficient animals. We demonstrate that increased accumulation of LPA in plaques induces perivascular mast cell activation and in this way contributes to plaque destabilization in vivo. This study points to local LPA availability as an important factor in atherosclerotic plaque stability.

Mast Cell Sarcoma with Megakaryocytic Differentiation in a Calf

A case of mast cell sarcoma in a 5-month-old Holstein female calf is described. Macroscopically, enlargement of the spleen, lymph nodes, tonsils and kidneys was noted, and there were tumor masses in the neck region and on the pleura and peritoneum. The pericardium and uterine and ureter walls were also involved by tumor. Most neoplastic cells had eosinophilic granules, which were metachromatic and positive for naphthol AS-D chloroacetate esterase and tryptase, whereas smaller numbers of cells were positive for factor VIII-related antigen, a marker of megakaryocytes. Some of the predominant type of these tumor cells were found within the epithelia of the lungs, tonsils, gastrointestinal tract, liver, ureters, urinary bladder and uterus. Their normal counterparts were considered to be globule leukocytes.

Sequential morphological characteristics of murine fetal liver hematopoietic microenvironment in Swiss Webster mice

Embryonic hematopoiesis occurs via dynamic development with cells migrating into various organs. Fetal liver is the main hematopoietic organ responsible for hematopoietic cell expansion during embryologic development. We describe the morphological sequential characteristics of murine fetal liver niches that favor the settlement and migration of hematopoietic cells from 12 days post-coitum (dpc) to 0 day post-partum. Liver sections were stained with hematoxylin and eosin, Lennert’s Giemsa, Sirius Red pH 10.2, Gomori’s Reticulin, and Periodic Acid Schiff/Alcian Blue pH 1.0 and pH 2.5 and were analyzed by bright-field microscopy. Indirect imunohistochemistry for fibronectin, matrix metalloproteinase-1 (MMP-1), and MMP-9 and histochemistry for naphthol AS-D chloroacetate esterase (NCAE) were analyzed by confocal microscopy. The results showed that fibronectin was related to the promotion of hepatocyte and trabecular differentiation; reticular fibers did not appear to participate in fetal hematopoiesis but contributed to the physical support of the liver after 18 dpc. During the immature phase, hepatocytes acted as the fundamental stroma for the erythroid lineage. The appearance of myeloid cells in the liver was related to perivascular and subcapsular collagen, and NCAE preceded MMP-1 expression in neutrophils, an occurrence that appeared to contribute to their liver evasion. Thus, the murine fetal liver during ontogenesis shows two different phases: one immature and mainly endodermic (<14 dpc) and the other more developed (endodermic-mesenchymal; >15 dpc) with the maturation of hepatocytes, a better definition of trabecular pattern, and an increase in the connective tissue in the capsule, portal spaces, and liver parenchyma. The decrease of hepatic hematopoiesis (migration) coincides with hepatic maturation.

Cytologic and Immunophenotypic Investigation of Lymphohematopoietic Neoplasms in Cattle

Ten cases of bovine lymphohematopoietic neoplasms were investigated histologically, histochemically, and immunohistochemically, and were classified into eight histologic types on the basis of the origin and morphology of tumor cells. Case 1, a precursor B lymphoblastic leukemia, was positive for CD79a and terminal deoxynucleotidyl transferase. Case 2, a thymic B cell lymphoma, was also positive for these markers, but there were cytologic differences between the two cases. Cases 3-5 were diagnosed as pleomorphic B cell lymphomas, which were characterized by cytologic pleomorphism and expression of CD79a and CD5, and were etiologically associated with bovine leukosis virus (BLV). A case of diffuse large B cell lymphoma of the cerebrum (case 6) also showed a positive result for CD79a and CD5. However, the lymphoma was composed of a homogeneous population of large neoplastic cells, and was considered to be unrelated to BLV. The other B cell cases were categorized into immunoblastic (case 7) and lymphoplasmacytic (case 8) lymphomas, in which immunoglobulin-producing lymphoma cells were observed. In a cutaneous γδ T cell lymphoma (case 9), the neoplastic cells cytologically resembled those in case 6, but expressed CD3 and WC1. In case 10, an acute basophilic leukemia, some leukemia cells had intracytoplasmic granules that were metachromatic and tryptase positive but negative for naphthol AS-D chloroacetate esterase. Bovine lymphohematopoietic malignancies are classifiable into discrete histologic types according to immunophenotype. The classification is more scientific than the traditional one, the latter being based on the age of affected animals and/or the site of tumor formation.

Acute Myeloid Leukemia with Mastocytic and Megakaryocytic Differentiation in a Calf

A diagnosis of acute myeloid leukemia was made in a 10-month-old Holstein female calf. The leukemia was macroscopically characterized by great enlargement of the spleen and moderate enlargement of some lymph nodes. Histochemical and immunohistochemical examination disclosed the presence of neoplastic cells either containing metachromatic and tryptase-positive granules or expressing factor VIII-related antigen. The granules, which were positive for naphthol AS-D chloroacetate esterase and did not have particulate contents, were distinct from those of basophilic leukemia cells. This leukemia was thought to be derived from a common myeloid progenitor capable of giving rise to megakaryocyte-erythrocyte progenitors and granulocyte-monocyte progenitors with the ability to differentiate into mast cells.

Haematology and leucocyte enzyme cytochemistry of a threatened yellow catfish Horabagrus brachysoma (Gunther 1864)

The haematology and leucocyte enzyme cytochemistry of Horabagrus brachysoma, a threatened freshwater catfish endemic to southern India, was studied using standard methods. Intra-specific variation was found for the haematological parameters, but this did not exceed the range of values observed in other catfishes. The relatively high haemoglobin (Hb) concentration may be indicative of an ability to breathe air and high activity. The erythrocytes are fully packed with Hb, revealing the bottom dwelling habit and primitive nature of this catfish. The leucocyte enzyme pattern also showed some variations from those of other fishes. Lymphocytes were positive only for peroxidase (PER) enzyme activity and negative for alkaline phosphatase (LAP), alpha-naphthyl acetate esterase (ANAE) and naphthol ASD chloroacetate esterase (ASDE). Monocytes were weakly positive for ANAE activity and negative for the other three enzymes tested. Neutrophils were negative for LAP, ANAE and ASDE but showed a moderately strong positive reaction for PER. Basophils and eosinophils were found to be devoid of all of these enzymes. Thrombocytes were observed to have weakly positive PER and ASDE, but there was no demonstrable LAP and ANAE activity. A number of characteristics were identified that distinguish this species from other fishes: (1) lymphocytes of H. brachysoma are actively engaged in both phagocytosis and defence mechanisms, while the monocytes participate in cellular defence mechanisms, primarily phagocytosis; (2) thrombocytes function as a protection barrier as well as carrying out their normal function of haemato plug formation during blood clotting. Results from the haematological and leucocyte cytochemical analyses reveal the haematological make-up and effective immune mechanism of this threatened fish and show it to be highly adaptive in nature. The data may be useful in programmes aiming the effective conservation of this species.

The inhibition of neutrophil elastase ameliorates mouse liver damage due to ischemia and reperfusion

Neutrophils are considered crucial effector cells in the pathophysiology of organ ischemia/reperfusion injury (IRI). Although neutrophil elastase (NE) accounts for a substantial portion of the neutrophil activity, the function of NE in liver IRI remains unclear. This study focuses on the role of NE in the mechanism of liver IRI. Partial warm ischemia was produced in the left and middle hepatic lobes of C57BL/6 mice for 90 minutes, and this was followed by 6 to 24 hours of reperfusion. Mice were treated with neutrophil elastase inhibitor (NEI; 2 mg/kg per os) at 60 minutes prior to the ischemia insult. NEI treatment significantly reduced serum alanine aminotransferase levels in comparison with controls. Histological examination of liver sections revealed that unlike in controls, NEI treatment ameliorated hepatocellular damage and decreased local neutrophil infiltration, as assessed by myeloperoxidase assay, naphthol AS-D chloroacetate esterase stains, and immunohistochemistry (anti-Ly-6G). The expression of pro-inflammatory cytokines (tumor necrosis factor alpha and interleukin 6) and chemokines [chemokine (C-X-C motif) ligand 1 (CXCL-1), CXCL-2, and CXCL-10] was significantly reduced in the NEI treatment group, along with diminished apoptosis, according to terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and caspase-3 activity. In addition, toll-like receptor 4 (TLR4) expression was diminished in NEI-pretreated livers, and this implies a putative role of NE in the TLR4 signal transduction pathway. Thus, targeting NE represents a useful approach for preventing liver IRI and hence expanding the organ donor pool and improving the overall success of liver transplantation. Liver Transpl 15:939-947, 2009. (c) 2009 AASLD.

Mast cell reactivity at the margin of human skin wounds: An early cell marker of wound survival?

Detecting the vitality of mechanical skin wounds (antemortem versus postmortem injury) in human cadavers remains a specifically forensic problem. To determine whether skin mast cells (MCs) are activated during the very early phase of human wound healing we performed a histomorphometric evaluation of the extent of MC enzyme loss as an indication of MC degranulation at the wound margins of skin wounds in 64 human cadavers. We compared the number of tryptase-reactive MCs, which are said not to loose all of their enzyme activity during degranulation process, with the number of naphthol AS-D chloroacetate esterase (NAS-DClAE)-positive MCs, which loose their complete enzyme activity in the form of enzyme-positive granula after activation. The enzyme activity was evaluated on sequential histological sections after autopsy as an indirect quantification of the number of degranulated MCs. Most of the victims had died within 10–60 min after injury ( n = 50), 12 survived between 60 min and 24 h, and only 2 victims survived more than 24 h (12 days each). The number of enzyme-positive MCs were counted in six successive visual fields (0.785 mm 2) on the one hand located parallel to and – on the other hand – at increasing distances outward from the wound margins. In victims surviving the injury less than 60 min the average number of NAS-DClAE-reactive MCs next to the wound margin was significantly lower than the number of tryptase-reactive MCs. The extent of the reduction in NAS-DClAE-reactive MC counts correlated inversely with the distance from the wound edges. Our findings show that MCs undergo very early loss of NAS-DClAE activity at wound margins, and thus appear to be an early cell marker of wound survival. However, definitive evidence that the enzyme loss (degranulation) represents a vital process can only be obtained by comparing MC enzyme loss induced by injury during intact circulation with the MC reaction to injury inflicted very shortly after cardiac arrest, a question that can only be answered by animal experiments.