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The Journal of Allergy and Clinical... May 2014Although several novel agents are currently in clinical trials for eosinophilic disorders, none has demonstrated efficacy in reducing blood and tissue eosinophilia in... (Clinical Trial)
Clinical Trial
The eosinophil surface receptor epidermal growth factor-like module containing mucin-like hormone receptor 1 (EMR1): a novel therapeutic target for eosinophilic disorders.
BACKGROUND
Although several novel agents are currently in clinical trials for eosinophilic disorders, none has demonstrated efficacy in reducing blood and tissue eosinophilia in all subjects. Additional approaches are clearly needed.
OBJECTIVE
We sought to explore the potential of the human eosinophil surface receptor epidermal growth factor-like module containing mucin-like hormone receptor 1 (EMR1) as a therapeutic target for eosinophilic disorders.
METHODS
EMR1 expression was assessed in blood and bone marrow specimens from eosinophilic and healthy subjects, cell lines, CD34(+) cells differentiated in vitro, and tissue biopsy specimens by using flow cytometry, quantitative PCR, and immunostaining. Eosinophil targeting by a novel, humanized, afucosylated anti-EMR1 IgG1 was evaluated in vitro by using a natural killer cell-mediated killing assay and in vivo in cynomolgus monkeys.
RESULTS
Analysis of blood and bone marrow cells from healthy and eosinophilic donors and in vitro-differentiated CD34(+) cells confirmed restriction of human EMR1 surface and mRNA expression to mature eosinophils. Tissue eosinophils also expressed EMR1. Although EMR1 was highly expressed on eosinophils from all subjects, surface expression was negatively correlated with absolute eosinophil counts (r = -0.46, P < .001), and soluble plasma levels correlated positively with absolute eosinophil counts (r = 0.69, P < .001), suggesting modulation of EMR1 in vivo. Nevertheless, afucosylated anti-EMR1 mAb dramatically enhanced natural killer cell-mediated killing of eosinophils from healthy and eosinophilic donors and induced a rapid and sustained depletion of eosinophils in monkeys.
CONCLUSION
EMR1 expression is restricted to mature blood and tissue eosinophils. Targeting of eosinophils with afucosylated anti-EMR1 antibody shows promise as a treatment for eosinophilic disorders.
Topics: Antibodies, Monoclonal, Murine-Derived; Bone Marrow Cells; CD4-Positive T-Lymphocytes; Calcium-Binding Proteins; Eosinophilia; Eosinophils; Female; Gene Expression Regulation; Humans; Immunoglobulin G; K562 Cells; Male; Membrane Glycoproteins; Mucins; Receptors, G-Protein-Coupled; U937 Cells
PubMed: 24530099
DOI: 10.1016/j.jaci.2013.11.041 -
Molecular Microbiology Feb 1999Prepilin peptidases cleave, among other substrates, the leader sequences from prepilin-like proteins that are required for type II protein secretion in Gram-negative...
Prepilin peptidases cleave, among other substrates, the leader sequences from prepilin-like proteins that are required for type II protein secretion in Gram-negative bacteria. To begin to assess the importance of type II secretion for the virulence of an intracellular pathogen, we examined the effect of inactivating the prepilin peptidase (pilD) gene of Legionella pneumophila. Although the pilD mutant and its parent grew similarly in bacteriological media, they did differ in colony attributes and recoverability from late stationary phase. Moreover, at least three proteins were absent from the mutant's supernatant, indicating that PilD is necessary for the secretion of Legionella proteins. The absence of both the major secreted protein and a haemolytic activity from the mutant signalled that the L. pneumophila zinc metalloprotease is excreted via type II secretion. Most interestingly, the pilD mutant was greatly impaired in its ability to grow within Hartmannella vermiformis amoebae and the human macrophage-like U937 cells. As reintroduction of pilD into the mutant restored inefectivity and as a mutant lacking type IV pilin replicated like wild type, these data suggested that the intracellular growth of L. pneumophila is promoted by proteins secreted via a type II pathway. Intratracheal inoculation of guinea pigs revealed that the LD50 for the pilD mutant is at least 100-fold greater than that for its parent, and the culturing of bacteria from infected animals showed a rapid clearance of the mutant from the lungs. This is the first study to indicate a role for PilD and type II secretion in intracellular parasitism.
Topics: Animals; Bacterial Proteins; Cell Survival; Colony Count, Microbial; Endopeptidases; Fimbriae, Bacterial; Guinea Pigs; Hartmannella; Humans; Legionella pneumophila; Lung; Microscopy, Electron; Mutagenesis; Proteins; Spleen; Statistics as Topic; Stem Cells; Temperature; Time Factors; U937 Cells
PubMed: 10048038
DOI: 10.1046/j.1365-2958.1999.01239.x -
The Journal of Experimental Medicine Nov 1975In a previous study also done with a liquid culture technique, the monoblast was identified and characterized as the most immature cell of the mononuclear phagocyte cell...
In a previous study also done with a liquid culture technique, the monoblast was identified and characterized as the most immature cell of the mononuclear phagocyte cell line recognized so far. The present study concerned the proliferative behavior of the monoblast and promonocyte in colonies. The cell-cycle times of both cell types were determined on the basis of four independent methods. The resulting values all show excellent agreement: for the monoblast 11.0-11.9 h, and for the promonocyte 11.4-12.8 h. The DNA-synthesis time found for the two cell types amounted to 5.7 h for the monoblast and 5.5 h for the promonocyte. The duration of the other phages of the cell cycle of the proliferating mononuclear phagocytes proved to be: G2 phase, 0.6 h; mitosis phage, 1.8 h; and G1 phase, 3.5-3.8 h. The individual colonies showed a biphasic pattern of colony growth, an initial phase of rapid proliferation being followed by a stage wtih a markedly decreased growth rate. In the initial stage only monoblasts are present in the colony; when the growth rate slows down promonocytes and macrophages appear. These observations support the earlier conclusion that the monoblast is without doubt the precursor of the promonycyte. Colony size was found to vary widely. The main factor underlying this variation proved to be the lag time between the start of the culture and the time point at which the colony-forming cells begin to divide. Mathematical analysis showed that the variation in colony size probably does not arise from heterogeneity of the population of colony-forming cells. A mathematical approach was used to determine the proportion of self-replicating and differentiating cells among the dividing monoblasts and promonocytes in the colony. The results indicate that initially in vitro the majority of the cells of both types are self-replicating cells, but later an increasing proportion of the dividing cells give rise to another, more mature type of cell. On the basis of the conclusion that the monoblast initiates the mononuclear phagocyte colony, the number of monoblasts (2.5 X 10(5)) present in vivo was estimated to be half the number of the promonocytes. In view of this ratio the mostly likely pattern for the proliferation of mononuclear phagocytes in the bone marrow is that a monoblast divides once, giving rise to two promonocytes which in their turn divide once and form two nonproliferating monocytes.
Topics: Animals; Bone Marrow Cells; Cell Count; Cell Differentiation; Cell Division; Cells, Cultured; Macrophages; Male; Mice; Monocytes; Phagocytes
PubMed: 1194851
DOI: 10.1084/jem.142.5.1200 -
Ultrastructural Pathology Apr 2013To describe characteristics of monocytes and histiocytes in the bone marrow of patients with a confirmed and suspected diagnosis of reactive histiocytosis.
OBJECTIVE
To describe characteristics of monocytes and histiocytes in the bone marrow of patients with a confirmed and suspected diagnosis of reactive histiocytosis.
METHODS
14 patients with a confident diagnosis of reactive histiocytosis or with a suspected diagnosis were inpatients at the Tianjin Blood Diseases Hospital between 2008 and 2012. Nucleated cells from bone marrow were observed by light microscopy - morphologically and immunohistochemically for histiocyte antigens - and ultrastructurally by transmission electron microscopy.
RESULTS
Monocytes, atypical histiocytes, macrophages, hemophagocytes, reticular cells and dendritic cells were significantly increased in 9, 9, 5, 3, 3 and 2, respectively, of the 14 cases. Atypical histiocytes expressed some morphological characteristics of promonocytes.
CONCLUSION
Monocytes, atypical histiocytes, macrophages, hemophagocytes, reticular cells and dendritic cells were increased in different relative degrees in patients with bone marrow reactive histiocytosis or suspected reactive histiocytosis. The increase in numbers of monocytes, atypical histiocytes and macrophages was a particularly significant feature. It is argued that atypical histiocytes with immature monocyte features might be precursors of hemophagocytes, reticular cells or dendritic cells.
Topics: Adolescent; Adult; Aged; Antigens, Differentiation; Bone Marrow; Bone Marrow Cells; Bone Marrow Examination; Cell Count; Child, Preschool; Dendritic Cells; Female; Fibroblasts; Histiocytes; Histiocytosis, Non-Langerhans-Cell; Humans; Infant; Male; Microscopy, Electron, Transmission; Monocytes; Phagocytes; Reticulocytes; Young Adult
PubMed: 23573889
DOI: 10.3109/01913123.2012.742174 -
Apoptosis : An International Journal on... Mar 2012The proto-oncogene, pleomorphic adenoma gene-like 2 (PLAGL2), is implicated in a variety of cancers including acute myeloid leukemia (AML), malignant glioma, colon...
The proto-oncogene, pleomorphic adenoma gene-like 2 (PLAGL2), is implicated in a variety of cancers including acute myeloid leukemia (AML), malignant glioma, colon cancer, and lung adenocarcinoma. There is additional evidence that PLAGL2 can function as a tumor suppressor by initiating cell cycle arrest and apoptosis. Interestingly, PLAGL2 has also been implicated in human myelodysplastic syndrome, a disease that is characterized by ineffective hematopoiesis and can lead to fatal cytopenias (low blood counts) as a result of increased apoptosis in the marrow, or, in about one-third of cases, can progress to AML. To gain a better understanding of the actions of PLAGL2 in human myeloid cells, we generated a stable PLAGL2-inducible cell line, using human promonocytic U937 cells. PLAGL2 expression inhibited cell proliferation which correlated with an accumulation of cells in G1, apoptotic DNA-laddering, an increase in caspase 3, 8, and 9 activity, and a loss of mitochondrial transmembrane potential. There was significant increase in the p53 homologue, p73, with PLAGL2 expression, and consistent with mechanisms of p73-regulated cell cycle control and apoptosis, there was increased expression of known p73 target genes p21, DR5, TRAIL, and Bax. PLAGL2-induced cell cycle block was abolished in the presence of p73 siRNA. Together, these data support a role for PLAGL2 in cell cycle regulation and apoptosis via activation of p73.
Topics: Apoptosis; Cell Cycle Checkpoints; Cell Division; Cell Line, Tumor; DNA-Binding Proteins; Gene Knockdown Techniques; HEK293 Cells; Humans; Nuclear Proteins; Proto-Oncogene Mas; RNA-Binding Proteins; Transcription Factors; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; U937 Cells
PubMed: 22076304
DOI: 10.1007/s10495-011-0672-3 -
Cell Biology International Jul 2007Under the influence of RANKL, in the presence of M-CSF, monocyte/macrophage precursor cells entered the osteoclast lineage and expressed the osteoclast marker...
Under the influence of RANKL, in the presence of M-CSF, monocyte/macrophage precursor cells entered the osteoclast lineage and expressed the osteoclast marker tartrate-resistant acid phosphatase (TRAP). These cells were motile and began to differentiate by contacting and fusing together, initially forming cells with several nuclei. All sizes of cells continued to fuse, forming larger cells with more than 6 and as many as 50 nuclei. The degree of osteoclastogenesis was related to the concentration of RANKL. High cell density changed osteoclast morphology from a more rounded form with cytoplasm extended all round the cell to a form with cytoplasm concentrated around the nuclei and more restricted multiple cytoplasmic extensions. At optimal cell density and RANKL concentrations the large numbers of rounded cells fused into large cytoplasmic masses. On reaching a critical size, osteoclasts assumed a spread morphology with a peripheral ring structure. Most of the nuclei were associated with the peripheral ring. When cytoplasmic masses were present, rings also formed within the mass, often with no contact with the cell periphery. All forms of RANKL-induced osteoclastogenesis were blocked by the endogenous decoy receptor osteoprotegerin and were also strongly reduced by calcitonin, with the later arriving morphological categories being the first to disappear.
Topics: Acid Phosphatase; Animals; Bone Marrow Cells; Cell Count; Cell Differentiation; Cell Size; Cells, Cultured; Dose-Response Relationship, Drug; Female; Isoenzymes; Mice; Mice, Inbred Strains; Osteoclasts; RANK Ligand; Tartrate-Resistant Acid Phosphatase
PubMed: 17303447
DOI: 10.1016/j.cellbi.2006.12.008 -
The Journal of Biological Chemistry Aug 2005The parasitic protozoan Leishmania specifically manipulates the expression of host macrophage genes during initial interactions, as revealed by mRNA differential display...
The parasitic protozoan Leishmania specifically manipulates the expression of host macrophage genes during initial interactions, as revealed by mRNA differential display reverse transcription-PCR and cDNA microarray analyses. The genes that are down-regulated in mouse (J774G8) or human (U937) macrophages upon exposure to Leishmania include small RNA transcripts from the short interspersed element sequences. Among the short interspersed element RNAs that are down-regulated is 7SL RNA, which is the RNA component of the signal recognition particle. Because the microbicidal functions of macrophages profoundly count on vesicular protein transport processes, down-regulation of 7SL RNA may be significant in the establishment of infection by Leishmania in macrophage phagolysosomes. To evaluate whether down-regulation of 7SL RNA results in inhibition of signal recognition particle-mediated vesicular protein transport processes, we have tested and found that the targeting of proteins to the endoplasmic reticulum and plasma membrane and the secretion of proteins by macrophages are compromised in Leishmania-infected J774G8 and U937 cells. Knocking down 7SL RNA using small interfering RNA mimicked the effect of exposure of macrophages to Leishmania. The overexpression of 7SL RNA in J774G8 or U937 cells made these cells resistant to Leishmania infection, suggesting the possible biological significance of down-regulation of 7SL RNA synthesis in the establishment of infection by Leishmania. We conclude that Leishmania down-regulates 7SL RNA in macrophages to manipulate the targeting of many proteins that use the vesicular transport pathway and thus favors its successful establishment of infection in macrophages.
Topics: Animals; Cell Differentiation; Cell Membrane; DNA, Complementary; Down-Regulation; Endoplasmic Reticulum; Gene Expression Profiling; Gene Expression Regulation; Humans; Leishmania; Leishmaniasis; Macrophages; Mice; Protein Transport; RNA; RNA, Messenger; RNA, Small Cytoplasmic; RNA, Small Interfering; Reverse Transcriptase Polymerase Chain Reaction; Signal Recognition Particle; Signal Transduction; Time Factors; Trypanosoma brucei brucei; U937 Cells; Up-Regulation
PubMed: 15955815
DOI: 10.1074/jbc.M504162200 -
Journal of Toxicology and Environmental... 2013This study detailed the sequence of recurring inflammatory events associated with episodic allergen exposures of mice resulting in airway hyperreactivity, sustained...
This study detailed the sequence of recurring inflammatory events associated with episodic allergen exposures of mice resulting in airway hyperreactivity, sustained inflammation, goblet-cell hyperplasia, and fibrogenesis that characterize a lung with chronic asthma. Ovalbumin (OVA)-sensitized female BALB/c mice were exposed to saline-control or OVA aerosols for 1 h per day for episodes of 3 d/wk for up to 8 wk. Lung inflammation was assessed by inflammatory cell recoveries using bronchoalveolar lavages (BAL) and tissue collagenase dispersions. Cell accumulations were observed within airway submucosal and associated perivascular spaces using immunohistochemical and tinctorial staining methods. Airway responsiveness to methacholine aerosols were elevated after 2 wk and further enhanced to a sustained level after wk 4 and 8. Although by wk 8 diminished OVA-induced accumulations of eosinophils, neutrophils, and monocyte-macrophages were observed, suggesting diminished responsiveness, the BAL recovery of lymphocytes remained elevated. Airway but not perivascular lesions persisted with a proliferating cell population, epithelial goblet-cell hyperplasia, and evidence of enhanced collagen deposition. Examination of lung inflammatory cell content before the onset of the first, second, and fourth OVA exposure episodes demonstrated enhancements in residual BAL lymphocyte and BAL and tissue eosinophil recoveries with each exposure episode. Although tissue monocyte-macrophage numbers returned to baseline prior to each exposure episode, the greatest level of accumulation was observed after wk 4. These results provide the basis for establishing the inflammatory and exposure criteria by which episodic environmental exposures to allergen might result in the development of a remodeled lung in asthma.
Topics: Aerosols; Allergens; Animals; Asthma; Bronchoalveolar Lavage Fluid; Cell Count; Chronic Disease; Collagen; Female; Fibrosis; Inhalation Exposure; Leukocytes; Lung; Methacholine Chloride; Mice; Mice, Inbred BALB C; Monocyte-Macrophage Precursor Cells; Ovalbumin; Recurrence; Respiratory Function Tests; Time Factors
PubMed: 23356647
DOI: 10.1080/15287394.2013.752323 -
Scandinavian Journal of Immunology Sep 2009HIV-infected individuals have an increased risk of invasive bacterial infections, even at early clinical stages with relatively normal CD4(+) T-cell counts. The...
HIV-infected individuals have an increased risk of invasive bacterial infections, even at early clinical stages with relatively normal CD4(+) T-cell counts. The pathogenic mechanisms behind this are not fully understood. However, an increasing number of studies indicate that HIV may impair the innate immunity to bacteria by infecting key cells of the monocyte/macrophage lineage. In this study, the effects of HIV infection on the protein profile of undifferentiated monocyte-like THP-1 cells were examined by a mass spectrometric approach based on stable isotope labelling with amino acid in cell culture (SILAC). We identified 651 proteins, of which nine proteins were down-regulated and 17 proteins were up-regulated in HIV-infected THP-1 cells as compared to uninfected controls. Most remarkably, the IL-1 receptor associated kinase 4 (IRAK-4), which is essential for virtually all TLR signalling, was suppressed, whereas the precursor for the antibiotic peptide Dermcidin was up-regulated in HIV-infected cells. Upon stimulation of either TLR2 or TLR4, the HIV-infected THP-1 cells displayed reduced TNF-alpha secretion. The HIV-induced down-regulation of IRAK-4 was reconfirmed in monocyte-derived macrophage cell cultures. These data suggests that HIV may impair the TLR signalling cascade for pathogen recognition in cells of the monocyte/macrophage lineage and thus, may reduce the ability of the innate immune system to sense invading pathogens and initiate appropriate responses.
Topics: Cell Line; Down-Regulation; HIV; HIV Infections; Humans; Immunity, Innate; Interleukin-1 Receptor-Associated Kinases; Macrophages; Monocytes; Peptides; Proteomics; Toll-Like Receptor 2; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha; Up-Regulation
PubMed: 19703016
DOI: 10.1111/j.1365-3083.2009.02299.x -
Clinical Cancer Research : An Official... Apr 2014Recent studies suggested that AKT activation might confer poor prognosis in acute myelogenous leukemia (AML), providing the rationale for therapeutic targeting of this...
PURPOSE
Recent studies suggested that AKT activation might confer poor prognosis in acute myelogenous leukemia (AML), providing the rationale for therapeutic targeting of this signaling pathway. We, therefore, explored the preclinical and clinical anti-AML activity of an oral AKT inhibitor, MK-2206. Experimental Methods: We first studied the effects of MK-2206 in human AML cell lines and primary AML specimens in vitro. Subsequently, we conducted a phase II trial of MK-2206 (200 mg weekly) in adults requiring second salvage therapy for relapsed/refractory AML, and assessed target inhibition via reverse phase protein array (RPPA).
RESULTS
In preclinical studies, MK-2206 dose-dependently inhibited growth and induced apoptosis in AML cell lines and primary AML blasts. We then treated 19 patients with MK-2206 but, among 18 evaluable participants, observed only 1 (95% confidence interval, 0%-17%) response (complete remission with incomplete platelet count recovery), leading to early study termination. The most common grade 3/4 drug-related toxicity was a pruritic rash in 6 of 18 patients. Nevertheless, despite the use of MK-2206 at maximum tolerated doses, RPPA analyses indicated only modest decreases in Ser473 AKT (median 28%; range, 12%-45%) and limited inhibition of downstream targets.
CONCLUSIONS
Although preclinical activity of MK-2206 can be demonstrated, this inhibitor has insufficient clinical antileukemia activity when given alone at tolerated doses, and alternative approaches to block AKT signaling should be explored.
Topics: Acute Disease; Administration, Oral; Adult; Aged; Aged, 80 and over; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Exanthema; Female; HL-60 Cells; Heterocyclic Compounds, 3-Ring; Humans; Immunoblotting; Leukemia, Myeloid; Male; Middle Aged; Proto-Oncogene Proteins c-akt; Pruritus; Salvage Therapy; Treatment Outcome; U937 Cells
PubMed: 24583795
DOI: 10.1158/1078-0432.CCR-13-1978