Nonspecific Esterase Staining Research Articles

AML with translocation t(8;16)(p11;p13) demonstrates unique cytomorphological, cytogenetic, molecular and prognostic features

Balanced chromosomal rearrangements define distinct entities in acute myeloid leukemia (AML). Here, we present 13 AML cases with t(8;16)(p11;p13) with observed low incidence (13/6124 patients), but more frequent presentation in therapy-related AML than in de novo AML (7/438 versus 6/5686, P=0.00001). Prognosis was poor with median overall survival of 4.7 months. Cytomorphology was characterized by parallel positive myeloperoxidase and non-specific esterase staining, therefore, French-American-British (FAB)-classification was impossible and origin of the AML with t(8;16) from an early stem cell with myeloid and monoblastic potential is hypothesized. Erythrophagocytosis was observed in 7/13 cases. Using gene expression profiling on 407 cases, patients with t(8;16) were compared to AML FAB subtypes with normal karyotype. Principal component analyses demonstrated that AML with t(8;16) were distinct from FAB subtypes M1, M4, M5a/b. When further compared to AML showing balanced rearrangements, that is, current WHO categories t(15;17), t(8;21), inv(16) and t(11q23)/MLL, AML with t(8;16) cases were clustered close to t(11q23)/MLL sharing commonly expressed genes. Subsequently, a pairwise comparison discriminated AML with t(8;16) from AML with t(11q23)/MLL, thus defining a highly unique signature for AML with t(8;16). In conclusion, AML with t(8;16) demonstrates unique cytomorphological, cytogenetic, molecular and prognostic features and is a specific subtype of AML.

An improved enrichment method for functionally competent, highly purified peripheral blood dendritic cells and its application to HIV-infected blood samples.

Dendritic cells (DC) were purified from human peripheral blood using a rapid and simple method based on magnetic depletion of phagocytes with carbonyl iron, followed by centrifugation of nonphagocytic cells on a Percoll density gradient and depletion of lymphocytes and macrophages/monocytes with a panel of MoAbs and immunomagnetic beads. Enriched DC were obtained with > 99% purity as judged by non-specific esterase (NSE) staining. After isolation, these cells, representing 0.4% of the starting mononuclear cell population, still function as potent antigen-presenting cells for purified T lymphocytes. The present results confirm the ability of human peripheral blood DC to present soluble antigens to T cells including microbial antigens and show, further, that DC are more potent soluble antigen-presenting cells than monocytes. The method was successfully applied to the purification of DC from the blood of HIV-infected individuals. We could not detect decreased numbers of DC in four individuals with early HIV infection and no replicating HIV was detected by in situ hybridization in the DC.

A Case of Atypical Canine Lymphoma with Oral Mass and Multiple Osteolysis

An 8-year-old female Golden Retriever had an oral mass and lameness. Multiple osteolysis of the systemic skeleton without monoclonal gammopathy was shown on electrophoresis of serum and urine samples. Cytological and histopathological examinations of the oral mass revealed atypical polymorphic cells similar to myeloid cells, and bone marrow aspiration indicated that these abnormal cells also might have invaded the bone marrow. These cells were negative to peroxidase and non-specific esterase staining, and clonal expansion of B lymphocytes could be detected by polymerase chain reaction (PCR) assay for antigen receptor gene rearrangement. The case was diagnosed as atypical lymphoma and treated by multi-drug chemotherapy. On the 142nd day after the first admission, the case had remission and the oral mass and multiple osteolysis were improved.

Can Exposure to Jet Fumes Lead to Acute Leukemia?.

Introduction:AML/MDS is usually diagnosed in older adults with median age of 64 in patients at time of diagnosis, after occupational exposure to organic solvents, such as benzene, or previous therapeutic exposure to alkylating agents, topoisomerase inhibitors, or ionizing radiation.We are presenting a case of AML/MDS in a young female without previous history of exposure to alkylating agents or ionizing radiation, but with history of exposure to jet fumes.Case Report:This is a 30-year old Carribean woman, chronic smoker, ethanol user, employed for eight years at the airport, working on the ground with heavy direct exposure to jet fumes, who presented with 2 weeks of progressive dyspnea, fatigue and bone pains. She was found to have pancytopenia, hemoglobin 2.8 gm/dL, platelets 37,000/mL, and neutrophils 1100/mL. Peripheral smear showed many nucleated red blood cells and blasts. Bone marrow aspiration was “dry” and bone biopsy revealed hypercellularity with dysplastic changes in erythrocyte, granulocyte and megakaryocytic lineages with 46% blasts. Flow cytometry of peripheral blood showed positivity for CD13, CD33, CD34, and CD117 and HLA-DR with complex cytogenetics showing deletions involving chromosomes 5q, 7q, 12p, 20q and loss of chromosome 17. Fluorescent in situ hybridization (FISH) studies were negative for t(15,17), MLL, t(8,21), inv(16) seen in classical de novo AML. FISH also did not show abnormalities in the probe regions 5q31, 7q31, 8cen and 20q12 of chromosomes 5, 7, 8 and 20 classically seen in de novo MDS. The blast cell morphology suggested acute myelocytic leukemia on a background of myelodysplasia. Precise FAB subtype was not possible in the absence of special stains like myeloperoxidase, and specific and non-specific esterase stains. The patient was treated with two induction courses of cytosine arabinoside and idarubicin. With persistence of 4.5% blasts on the peripheral blood and severe bone marrow aplasia, she was referred for allogeneic stem cell transplantation, in view of failed induction remission and complex cytogenetics at presentation.Conclusion:Case-control studies of leukemia demonstrated only slight increase in risk of disease after many years of occupational or chemical exposures. More studies to investigate leukemia incidence in airport employees, or even communities near airports are needed.

Mac-1 and F4/80 Surface Markers Are Present on Murine Hematopoietic Stem Cells.

Transplanted bone marrow donor cells with tissue specific phenotypes have been found in the brain, liver, heart, skin, lung, kidney, and gut of transplanted humans and mice. Such observations have led to the controversial hypothesis that hematopoietic stem cells (HSC) might be intrinsically plastic, and through transdifferentiation or fusion lead to the repair of damaged tissues throughout the body. Alternately, it is suggested that fusion of macrophages to the recipient cells may explain this phenomenon. We have shown recently that purified HSC are the cells responsible for GFP positive donor-derived muscle fibers in the recipient mice post bone marrow transplantation. However, further studies sorting for macrophage markers Mac-1 and F4/80 also resulted in donor-derived muscle fibers in the host. To address this discrepancy, we investigated subpopulations of Mac-1 and F4/80 positive cells, in the presence or absence of stem cell markers (Sca-1 and C-kit). We demonstrate that only the subpopulations of Mac-1 and F4/80 positive cells harboring stem cell markers, Sca-1 or c-kit, were capable of contributing to the regenerating muscle post transplantation. Furthermore, these same subpopulations demonstrated single cell High Proliferative Potential (HPP) (6–26%) in a 7 factor cytokine cocktail, compared to the Mac-1 or F4/80 cells with no stem cell markers (0%). Additionally, they demonstrated long-term engraftment in all three lineages at 1-year (average chimerism of 55% versus 0% in stem cell marker negative groups). These subpopulations were also evaluated for morphology using Hematoxylin/Eosin (H/E), Wright-Giemsa, and Nonspecific Esterase staining. In the Mac-1 and F4/80 positive groups, those negative for stem cell markers resembled differentiated cells of the myeloid origin (macrophages, granulocytes), while those with positive stem cell markers demonstrated stem cell characteristics. We did not observe any engraftability, donor-derived muscle fibers, or HPP potential for CD14 or cfms positive cells coexpressing stem cell markers, indicating that these markers are more appropriate for identifying macrophages. In conclusion, our studies demonstrate that both Mac-1 and F4/80 surface markers are present on HSC and therefore caution must be taken in the interpretation of data using these macrophage markers. It is reasonable to believe that the use of Mac-1 and/or F4/80 surface markers in a lineage depletion process may result in the loss of a subpopulation of stem cells, and other markers such as CD14 or c-fms may be more appropriate for eliminating differentiated macrophages.

Immunopathological changes of micro-vessels in dermatomyositis

To investigate the immunopathological changes of micro-vessels in dermatomyositis. Twelve patients with dermatomyositis, 5 males and 7 females, aged 40.8 (6 - 72), underwent muscle biopsy of biceps muscle of arm (n = 11) or deltoid muscle (n = 1). The specimens underwent routine histological, enzyme histochemical, and immunohistochemical staining and microscopy. Ten biopsy specimens from patients with other diseases were used as controls. Perifascicular atrophy of muscular fibers and inflammatory infiltration in perimysium were seen in all patients with dermatomyositis. Non-specific esterase staining showed deep staining of capillaries and micro vascular endothelium among the muscular fibers. Immunohistochemistry showed remarkable reduction of capillaries positive in von Willebrand factor (vWF), thrombomodulin (TM), and endothelial cell nitric oxide synthase (eNOS) in the perifascicular region, and low expression of .eNOS and TM in the microvascular endothelium in the perimysium. The vascular lesions of dermatomyositis are located not only in capillaries, but also in other microvessels. Lower expression of TM and eNOS in vascular endothelium suggests the reduction of anticoagulation and vasodilation functions of vascular endothelium. Dermatomyositis is an inflammatory vascular endothelial disease.

The efficient isolation of murine splenic dendritic cells and their cytochemical features

Despite their importance in professional antigen presentation and their ubiquitous presence, dendritic cells (DCs) are usually found in such trace amounts in tissues that their isolation with high purity is a difficult task. Because of their scarcity, accurate determination of the purity of isolated dendritic cells is very important. In this study, we purified murine splenic dendritic cells by a three-step enrichment method and evaluated their morphological, cytochemical and functional characteristics. Purity of the isolated cells was determined by established methods such as flow cytometry (FC) and immunocytochemistry (ICC) using anti-CD11c monoclonal antibody. In order to test purified DC functional properties, we used in vivo antigen presentation assay. Our results showed that antigen-pulsed DCs are potent stimulators of antigen-specific lymphocyte proliferation. We studied myeloperoxidase (MPO) and non-specific esterase (NSE) activity in isolated cells to determine the purity of dendritic cells compared to more conventional methods. Our results showed that murine splenic dendritic cells were deficient in both MPO and NSE activity and the percentage of purity obtained by NSE staining on isolated cells was comparable to the results obtained by either FC or ICC. To our knowledge, this is the first report on using NSE activity for determination of the purity of isolated murine splenic dendritic cells. We, therefore, recommend that NSE activity be employed as a simple, inexpensive and yet accurate method for evaluation of the purity of isolated murine splenic dendritic cells.

Mitochondrial gene knockout HL60ρ 0 cells show preferential differentiation into monocytes/macrophages

This study compares the differentiation potential of mitochondrial gene knockout (ρ 0) and parental HL60 cells in response to 1.25% dimethylsulfoxide (DMSO) and 10 nM phorbol myristate acetate (PMA). Compared to HL60 cells, undifferentiated HL60ρ 0 cells showed partial monocyte/macrophage differentiation, with increased CD11c and CD14 expression, decreased CD71 expression, and weak non-specific esterase staining. Differentiation along the monocyte/macrophage pathway (PMA) was more pronounced in HL60ρ 0 than parental HL60 cells with increased CD11c and CD14 expression and stronger non-specific esterase staining. DMSO-exposure resulted in a poorly differentiated nuclear morphology, small respiratory burst and marginal up-regulation of CD15 expression in HL60ρ 0 cells.

Peritoneal fluid cytokines and sICAM-1 in minimal endometriosis: search for discriminating factors between infertility and/or endometriosis

Objective:To evaluate cytokine levels (IL-1β, TNF-α, IL-6, IL-8), soluble intercellular adhesion molecule-1 (sICAM-1) and number of macrophages in peritoneal fluid (PF) of women with no minimal endometriosis and associated (or not) infertility in order to discriminate between infertility and/or endometriosis. Study design:Cytokines and sICAM-1 were measured by using quantitative enzyme-linked immunosorbent assay (ELISA) while the macrophages were identified by May-Grunwald-Giemsa and non-specific esterase staining and presented as medians. The measurements were performed in 78 women belonging to four selected subgroups according to their endometriosis and/or infertility status. Statistical analysis was performed using Kruskal–Wallis non-parametric ANOVA test. Additionally, discriminant function analyses were performed. Results:We have found the most elevated macrophage numbers in the groups of women with endometriosis. IL-1β did not present any statistically significant values differentiating the analysed subgroups. IL-6 (110.0pg/ml) and TNF-α exhibited the highest concentrations (statistically significant) in a group of fertile women with endometriosis. IL-8 clearly differentiated between the subgroups with infertility and sICAM-1 was statistically significantly elevated in the subgroups of infertile women. In the forward discriminant analysis, when subdividing the studied population according to its infertility status (we considered macrophages, IL-8 and IL-6 in order of probability values), we have found striking probability value of 92% for the correct classification into an infertile population. Conclusion:Out of the range of the analysed factors we have found only the sICAM-1 to be singled out between the standard discriminant analysis and the forward one. However, this important flagging molecule might be of considerable value for discrimination between different types of pathology at the level of immune effector function. The increased levels of TNF-α and IL-6 signified a group of fertile women with endometriosis; however only IL-6 presented a discriminating value in multifunctional analysis of examined subgroups. The analysed range of factors had a greater tendency to discriminate between infertility status rather than endometriosis.

Microarray Analysis of the Short Isoform of Ikaros Overexpressed in Patients with Hematologic Malignancies.

We analyzed expression of the transcription factor Ikaros in human hematologic malignancies and found overexpression of the short isoform of Ikaros, Ik-6, in patients with blast crisis of chronic myeloid leukemia and pre-B cell acute lymphoblastic leukemia. We have made several stable transfectants which overexpress Ik-6 and analyzed the consequence of Ik-6 overexpression.First, overexpression of Ik-6 in FDH-1, which was established from a patient with early pre-B acute lymphoblastic leukemia, and Ramos conferred resistance to dexamethasone- and anti-IgM-induced apoptosis, respectively. Upon treatment with dexamethasone, FDH-1 cells demonstrated massive apoptosis measured by staining with Annexin V. In contrast to mock transfectants, Ik-6 transfectants showed less marked apoptosis (mock 86.4% vs Ik-6 34.4–73.6%, p=0.01). We also used Ramos as a model of anti-IgM-induced apoptosis, and flow cytometric analysis was performed with Annexin-V staining. In contrast to mock transfectants, Ik-6 transfectants showed less pronounced apoptosis (mock 40.4% vs Ik-6 23.1–27.4%, p=0.03). Next, overexpression of Ik-6 in Nalm6 lead to impaired cell growth and cell cycle progression. Cell growth and cell cycle were analyzed and compared with mock transfectants and Ik-6 transfectants using MTT assay and DNA content analysis with PI staining, respectively. MTT assay demonstrated that, after three days of growth, Ik-6 transfectants grew more slowly than mock transfectants (Ik-6/mock ratio 0.62–0.67; p=0.01). DNA content analysis showed that, after three days of growth, more Ik-6 transfectants underwent G0–G1 arrest than mock transfectants (Ik-6/mock ratio 1.15–1.21; p=0.01), and less Ik-6 transfectants entered into S phase than mock transfectants (Ik-6/mock ratio 0.84–0.90; p=0.05).Finally, overexpression of Ik-6 promoted all-trans retinoic acid (ATRA)-induced granulocytic differentiation, and, on the other hand, inhibited vitamin D3 (VD3)-induced monocytic differentiation of HL-60. Upon induction of granulocytic differentiation by ATRA, mock transfectants showed 74.5% positivity of myeloperoxidase (MPO) staining. In contrast, Ik-6 transfectants showed 51.5–56.8% of positivity of MPO staining (p=0.04). NBT reduction assay demonstrated 43.8% positivity with mock transfectants and 62.3–67.3% positivity with Ik-6 transfectants (p=0.001). Upon induction of monocytic differentiation by VD3, mock transfectants showed 51.0% positivity of nonspecific esterase (NSE) staining. In contrast, Ik-6 transfectants showed 26.8–36.0% of positivity of NSE staining (p=0.03).To understand the underlying mechanisms, we report the results of microarray analysis of Ik-6 transfectants.

HIV-1 Protease Inhibitor Ritonavir Potentiates the Effect of 1,25-Dihydroxyvitamin D3 to Induce Growth Arrest and Differentiation of Human Myeloid Leukemia Cells Via Inhibition of CYP24.

Ritonavir (RTV) is a potent inhibitor of cytochrome p450 (CYPs) enzymes. This study explores the effects of RTV on CYP24 which converts 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] to its inactive form 1,24,25,(OH)3. Real-time RT-PCR showed that exposure of HL-60 cells to 1,25(OH)2D3 (10−7 M, 1h) induced expression of CYP24 by 80-fold, and pre-incubation of these cells with RTV (2X10−5 M, 3h) decreased 1,25(OH)2D3-stumulated expression of CYP24 transcripts by 30 %, resulting in increased levels of 1,25(OH)2D3 in culture media (50.7 vs 82.4 nmol/L). Importantly, pre-incubation of HL-60 cells with RTV (2X10−5 M, 3h) and then exposure to 1,25(OH)2D3 (10−7 M, 24 h) also increased intracellular levels of 1,25(OH)2D3 (1.20 vs 2.46 nmol/L) and potentiated the ability of 1,25(OH)2D3 to induce growth arrest of HL-60 cells. Clonogenic assay showed that either RTV (5X10−6 M) or 1,25(OH)2D3 (10−8 M) alone inhibited colonal growth of HL-60 cells by either a mean 30 ± 12 (±SD) % and 40 ± 12 %, respectively; when both were combined, colony formation was inhibited by a mean 80 ± 13 % (p<0.01). Measurement of CD14 expression, NBT reduction, and non-specific esterase staining showed that RTV augmented the prodifferentiative effects of 1,25(OH)2D3 in HL-60 cell. For example, 1,25(OH)2D3 (10−8 M, 2 days) induced approximately 48 % of HL-60 cells to become CD14 antigen positive. RTV (2X10−5 M, 48h) alone did not stimulate expression of CD14 antigen; when 1,25(OH)2D3 was combined with RTV, approximately 65 % of cells expressed CD14. Furthermore, androgen-independent DU145 human prostate cancer cells, that are resistant to growth inhibition by 1,25(OH)2D3, expressed 80-fold higher levels of CYP24 transcripts compared to HL-60 cells, and intracellular levels of 1,25(OH)2D3 were not detectable (<0.3 nmol/L) after exposure of these cells to 1,25(OH)2D3 (10−7 M, 24 h). Of note, pre-incubation of these cells with RTV (2X10−5 M, 3h) and then exposure to 1,25(OH)2D3 (10−7 M, 24 h) increased intracellular levels of 1,25(OH)2D3 to 0.94 nmol/L and sensitized DU145 cells to growth inhibition mediated by 1,25(OH)2D3 as measured by MTT assay. Taken together, inhibition of CYP24 might open a new paradigm for therapy using vitamin D compounds.

325 Drug interactions with neoplastic agents

A modified ‘low pH’ lignocaine method for the recovery of human monocytes from untreated plastics is described and compared with other adherence separation procedures, viz. mechanical scraping by a rubber policeman, microexudate-coated plastic method and pretreatment of plastics by fetal calf serum (FCS).Monocytes separated by each of the above-mentioned techniques carried out in parallel were characterised by morphological and functional criteria: non-specific esterase staining, contamination by T- and B-lymphocytes, viability, adherence, neutral red uptake, phagocytosis of yeast particles, random mobility, chemotaxis, and tumoricidal activity both in the absence and in the presence of interferon and lymphokines.The ‘low pH’ lignocaine method appears to be a simple and reproducible technique for the isolation of human monocytes. It seems preferable to other adherence procedures inasmuch as it yields viable, pure and functionally intact monocytes without requiring preconditional plastics.

324 Germ cell tumours

A modified ‘low pH’ lignocaine method for the recovery of human monocytes from untreated plastics is described and compared with other adherence separation procedures, viz. mechanical scraping by a rubber policeman, microexudate-coated plastic method and pretreatment of plastics by fetal calf serum (FCS).Monocytes separated by each of the above-mentioned techniques carried out in parallel were characterised by morphological and functional criteria: non-specific esterase staining, contamination by T- and B-lymphocytes, viability, adherence, neutral red uptake, phagocytosis of yeast particles, random mobility, chemotaxis, and tumoricidal activity both in the absence and in the presence of interferon and lymphokines.The ‘low pH’ lignocaine method appears to be a simple and reproducible technique for the isolation of human monocytes. It seems preferable to other adherence procedures inasmuch as it yields viable, pure and functionally intact monocytes without requiring preconditional plastics.

Two histone deacetylase inhibitors, trichostatin A and sodium butyrate, suppress differentiation into osteoclasts but not into macrophages

Histone deacetylase (HDAC) inhibitors are emerging as a new class of anticancer therapeutic agents and have been demonstrated to induce differentiation in some myeloid leukemia cell lines. In this study, we show that HDAC inhibitors have a novel action on osteoclast differentiation. The effect of 2 HDAC inhibitors, trichostatin A (TSA) and sodium butyrate (NaB), on osteoclastogenesis was investigated using rat and mouse bone marrow cultures and a murine macrophage cell line RAW264. Both TSA and NaB inhibited the formation of preosteoclast-like cells (POCs) and multinucleated osteoclast-like cells (MNCs) in rat bone marrow culture. By reverse transcription-polymerase chain reaction analysis, TSA reduced osteoclast-specific mRNA expression of cathepsin K and calcitonin receptor (CTR). In contrast, TSA and NaB did not affect the formation of bone marrow macrophages (BMMs) induced by macrophage colony-stimulating factor as examined by nonspecific esterase staining. Fluorescence-activated cell sorting analysis showed that TSA did not affect the surface expression of macrophage markers for CD11b and F4/80 of BMMs. TSA and NaB also inhibited osteoclast formation and osteoclast-specific mRNA expression in RAW264 cells stimulated with receptor activator of nuclear factor-kappa B (NF-kappa B) ligand (RANKL). Transient transfection assay revealed that TSA and NaB dose dependently reduced the sRANKL-stimulated or tumor necrosis factor alpha (TNF-alpha)-stimulated transactivation of NF-kappa B-dependent reporter genes. The treatment of RAW264 cells with TSA and NaB inhibited TNF-alpha-induced nuclear translocation of NF-kappa B and sRANKL-induced activation of p38 mitogen-activated protein kinase (MAPK) signals. These data suggest that both TSA and NaB exert their inhibitory effects by modulating osteoclast-specific signals and that HDAC activity regulates the process of osteoclastogenesis.

Combination of 22-oxa-1,25-dihydroxyvitamin D(3), a vitamin D(3) derivative, with vitamin K(2) (VK2) synergistically enhances cell differentiation but suppresses VK2-inducing apoptosis in HL-60 cells.

We originally reported that vitamin K(2) (VK2) effectively induces apoptosis in various types of primary cultured leukemia cells and leukemia cell lines in vitro. In addition, VK2 was shown to induce differentiation of leukemia cells when the cells were resistant against VK2-inducing apoptosis. A novel synthetic vitamin D(3)derivative, 22-oxa-1,25-dihydroxyvitamin D(3) (OCT: oxacarcitriol) shows a more potent differentiation-inducing ability among myeloid leukemia cells in vitro with much lesser extent of the induction of hypercalcemia in vivo as compared to the effects of 1alpha,25(OH)(2)D(3). In the present study, we focused on the effects of a combination of OCT plus VK2 on leukemia cells. Treatment of HL-60 cells with OCT for 72 h induces monocytic differentiation. A combination of OCT plus VK2 dramatically enhances monocytic differentiation as assessed by morphologic features, positivity for non-specific esterase staining, and cell surface antigen expressions. This combined effect far exceeds the maximum differentiation induction ability at the optimal concentrations of either OCT or VK2 alone. In addition, pronounced accumulation of the cells in the G0/G1 phase is observed by combined treatment with OCT plus VK2 as compared with each vitamin alone. In contrast to cell differentiation, caspase-3 activation and apoptosis induction in response to VK2 are significantly suppressed in the presence of OCT in HL-60 cells. These data suggest that monocytic differentiation and apoptosis induction of HL-60 cells are inversely regulated. Furthermore, pronounced induction of differentiation by combined treatment with VK2 plus OCT was also observed in four out of six cases of primary cultured acute myeloid leukemia cells in vitro, suggesting that VK2 plus OCT might be a potent combination for the differentiation-based therapy for acute myeloid leukemias.

Importance of MEK-1/-2 signaling in monocytic and granulocytic differentiation of myeloid cell lines

Activation of the MEK/ERK/MAP kinase signaling pathway promotes the proliferation and survival of hematopoietic cells. The kinases MEK-1, MEK-2, ERK-1/MAPK and ERK-2/MAPK are activated by phosphorylation at specific sites, and these events can be monitored using phospho-specific antibodies. In this report we examined the importance of the MEK/ERK/MAP kinase pathway in the monocytic and granulocytic differentiation of myeloid cell lines. Induction of monocytic differentiation in HL-60 cells by treatment with phorbol 12-myristate 13-acetate (PMA) led to rapid and sustained activation of MEK-1/-2, ERK-1/MAPK and ERK-2/MAPK, while induction of granulocytic differentiation by retinoic acid (RA) caused similar activation of MEK-1/-2 and ERK-2/MAPK, but not ERK-1/MAPK. The total levels of these kinases were not affected during the course of differentiation along either pathway. Pretreatment of cells with 5 microM of the MEK-1/-2-specific inhibitor U0126 abrogated PMA- or RA-induced activation of ERK-1/MAPK and ERK-2/MAPK. Importantly, pretreatment of HL-60 cells with U0126 was found to potently inhibit both monocytic and granulocytic differentiation, as assessed by cytochemical staining for non-specific esterase or nitroblue tetrazolium reduction, flow cytometric analysis of myeloid surface markers, and immunoblotting for the cell cycle inhibitor p21 WAF1/Cip1. Similar results were seen in U937 cells, where U0126 inhibited PMA-induced monocytic differentiation, and in 32D cells, where G-CSF-induced granulocytic differentiation was inhibited by U0126 pretreatment. Additional experiments revealed that inhibition of MEK-1/-2 in HL-60 cells resulted in nearly complete inhibition of differentiation-induced cell death during monocytic differentiation. By contrast, U0126 only partially inhibited cell death resulting from granulocytic differentiation. Taken together, our findings demonstrate that the MEK/ERK/MAP kinase signaling pathway is activated, and plays a critical role, during both monocytic and granulocytic differentiation of myeloid cell lines.

The Use of Enzyme Histochemistry in Detecting Cutaneous Toxicity of Three Topically Applied Jet Fuel Mdttures

The purpose of this study was to assess cutaneous enzyme activity in response to topical exposure to jet fuel, using enzyme histochemistry to analyze alkaline phosphatase (ALP), acid phosphatase (ACP), and nonspecific esterase (NSE) in the skin of pigs. Yorkshire pigs were exposed to Jet-A, JP-8, and JP-8(100) fuels for 5 h, 24 h, and 5 days under occluded Hill Top chambers (25 μL), nonoccluded conditions (25 μL), and occluded fabric conditions (335 μL). For ALP, the fabric Jet-A, JP-8, and JP-8(100) were significantly more intense (p < .05) than control sites in the stratum basale layer. ACP staining showed a general increase between 24 h and 5 days in most layers in the Hill Top chamber experiment, but no treatment effects. The sites of nonoccluded fabric involving JP-8 and JP-8(100) demonstrated significantly more staining (p < .05) of NSE than at control sites at 24 h in both the stratum spinosum and stratum basale layers. In addition, NSE staining at 5 days at the site where the fabric was dosed with JP-8 and JP-8(100) was significantly greater (p < .05) than in the occluded-fabric control site in the stratum granulosum layer. The increased distribution and significant difference in staining by these enzymes in jet fuel-treated skin across many layers (particularly ALP) strongly supports the conclusion that enzyme histochemistry can be used effectively as an early biomarker of cellular injury.

Abundance of Siderophages in Sputum

To investigate the lung response to traffic-related air pollution by enumerating hemosiderin-laden alveolar macrophages (AM) in sputum. Sputum samples were collected from 103 urban adult males from Calcutta chronically exposed to automobile exhaust. Forty-nine rural individuals served as controls. AM were identified by nonspecific esterase staining. Perl's Prussian blue technique was employed for the detection of hemosiderin-laden AM (siderophages). The urban group, consisting of 31 traffic officers, 25 automobile service station workers and 47 street hawkers, had seven times more AM in their sputum than did the matched controls. Besides, a remarkable rise (27-fold) in the number of siderophages in sputum was observed in urban individuals. Smoking further elevated the AM count and number of siderophages. Abundant siderophages in the urban group may indicate the toxic effect of airborne pollutants on the lung, leading to phagocytosis of destroyed cells, including erythrocytes, and accumulation of iron in AM. Enumeration of siderophages in sputum appears to be a simple, noninvasive, inexpensive cytochemical technique well suited to preliminary assessment of the adverse effects of air pollution on the lungs in large, population-based studies, especially in developing countries.

Mammalian Peptidoglycan Recognition Protein Binds Peptidoglycan with High Affinity, Is Expressed in Neutrophils, and Inhibits Bacterial Growth

Peptidoglycan recognition protein (PGRP) is conserved from insects to mammals. In insects, PGRP recognizes bacterial cell wall peptidoglycan (PGN) and activates prophenoloxidase cascade, a part of the insect antimicrobial defense system. Because mammals do not have the prophenoloxidase cascade, its function in mammals is unknown. However, it was suggested that an identical protein (Tag7) was a tumor necrosis factor-like cytokine. Therefore, the aim of this study was to identify the function of PGRP in mammals. Mouse PGRP bound to PGN with fast kinetics and nanomolar affinity (K(d) = 13 nm). The binding was specific for polymeric PGN or Gram-positive bacteria with unmodified PGN, and PGRP did not bind to other cell wall components or Gram-negative bacteria. PGRP mRNA and protein were expressed in neutrophils and bone marrow cells, but not in spleen cells, mononuclear cells, T or B lymphocytes, NK cells, thymocytes, monocytes, and macrophages. PGRP was not a PGN-lytic or a bacteriolytic enzyme, but it inhibited the growth of Gram-positive but not Gram-negative bacteria. PGRP inhibited phagocytosis of Gram-positive bacteria by macrophages, induction of oxidative burst by Gram-positive bacteria in neutrophils, and induction of cytokine production by PGN in macrophages. PGRP had no tumor necrosis factor-like cytotoxicity for mammalian cells, and it was not chemotactic on its own or in combination with PGN. Therefore, mammalian PGRP binds to PGN and Gram-positive bacteria with nanomolar affinity, is expressed in neutrophils, and inhibits growth of bacteria.

Expression of Nonspecific Esterase (NSE) by Thyroid Follicular Epithelium as a Marker for the Target Organ Susceptibility to Immune System Attack

Spontaneous autoimmune thyroiditis in obese strain (OS) chickens provides an excellent animal model for the study of Hashimoto's autoimmune thyroiditis in humans. The data presented in this paper indicate that nonspecific esterases (NSE) may play a role in or serve as a marker for the target organ susceptibility. Experiments have shown that follicular epithelial cells and interfollicular macrophages in connective tissue stain positively for NSE as early as the first day after hatching, a time at which infiltrating lymphocytes are not yet observed. We also have observed NSE positivity of follicular cells in the vicinity of mononuclear cell infiltration in all OS chickens, as well as weaker positivity in 6-month-old, avian leukosis virus free, Brown Leghorn outbred chickens, which appears in each case to correlate with infiltration of lymphocytes. In F 2 hybrids between OS and healthy CB inbred chickens, the intensity of NSE staining was more variable than in OS chickens. Using specific inhibitors eserine, Na-taurocholat and p-hydroxymercuribenzoic acid, we were able to inhibit in vitro the NSE positivity of thyroid gland follicular epithelium, indicating that this staining was not an artifact. Experiments are currently in progress to clarify the relationship between the presence of NSE in follicular epithelium and the predisposition to spontaneous autoimmune thyroiditis.