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The polymerase chain reaction technique (PCR), a primer-mediated enzymatic amplification of specific target sequences, was used to monitor gene transfer into hematopoietic progenitor stem cells. A gene coding for human interleukin 1 alpha... more
The polymerase chain reaction technique (PCR), a primer-mediated enzymatic amplification of specific target sequences, was used to monitor gene transfer into hematopoietic progenitor stem cells. A gene coding for human interleukin 1 alpha (IL-1 alpha) was cotransfected with the rous sarcoma virus (RSV) CAT plasmid into mouse bone marrow cells by electroporation. Individual chloramphenicol (CAM)-resistant bone marrow progenitor colonies (granulocyte-macrophage colony-forming units; CFU-GM) containing 50-100 cells were analyzed by PCR for the presence and expression of IL-1 alpha and CAT sequences. Amplified IL-1 alpha DNA sequences were detected from a 50-cell CFU-GM colony. CAT and IL-1 alpha RNA expression was demonstrated from the CAM-resistant CFU-GM colonies.
Research Interests:
Research Interests:
Leukotrienes are vasoactive arachidonic acid metabolites which are released by mast cells during hypersensitivity reactions. The mechanisms for regulating leukotriene biosynthesis are not well understood. A murine mast/basophil cell line... more
Leukotrienes are vasoactive arachidonic acid metabolites which are released by mast cells during hypersensitivity reactions. The mechanisms for regulating leukotriene biosynthesis are not well understood. A murine mast/basophil cell line (PT-18) was used to investigate this problem. Exogenously supplied [14C]arachidonic acid is not appreciably converted to leukotrienes by untreated PT-18 cells. However, when the cells were preincubated with the lymphocyte product 15-hydroxyeicosatetraenoic acid (15-HETE), addition of [14C]arachidonic acid consistently resulted in a dose-dependent synthesis of large amounts of both [14C]leukotriene B4 and [14C]5-HETE. These metabolites were isolated by high pressure liquid chromatography, converted to the methyl ester trimethylsilyl ether derivatives, and the structures confirmed by gas chromatography-mass spectrometry. These findings indicate that 15-HETE induces a direct activation of a cryptic 5-lipoxygenase in these cells. The closely related 12-HETE was ineffective. The activation phenomenon occurs rapidly and is reversible. Furthermore, the activation appears to be highly cell- and enzyme-specific, since lipoxygenases in three primary cell types including one that contains a 5-lipoxygenase and six other cell lines did not show this specific induction of leukotriene biosynthesis by 15-HETE. This report is the first evidence that 15-HETE, a major arachidonate metabolite in lymphocytes, can act as a signal to activate leukotriene production by susceptible mast cells.
Research Interests: Chemistry, Biology, Mast Cells, Biological Chemistry, Medicine, and 14 moreBiological Sciences, Tissue culture, Mice, Animals, Biological, High Pressure Liquid Chromatography, CHEMICAL SCIENCES, Biosynthesis, Arachidonic Acid, Basophil, basophils, Strains, Medical and Health Sciences, and leukotriene
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Research Interests:
Research Interests:
Research Interests:
Research Interests:
N-Acyl 4-(5-pyrimidine-2,4-dionyl)phenylalanine derivatives of type 4 were designed to replace the 2,6-dichlorobenzoylamine portion of compound 1 in order to identify novel compounds with improved potency against α4-integrins. Several... more
N-Acyl 4-(5-pyrimidine-2,4-dionyl)phenylalanine derivatives of type 4 were designed to replace the 2,6-dichlorobenzoylamine portion of compound 1 in order to identify novel compounds with improved potency against α4-integrins. Several derivatives were identified as very potent dual-acting α4-integrin, α4β1 and α4β7 antagonists. Investigation of a limited number of prodrug esters led to the discovery of the ethyl ester prodrug 42, which demonstrated good intestinal fluid stability and good permeability. Despite low solubility, 42 gave acceptable blood levels of 30 when dosed orally in non-human primates. Additionally, 42 had an overall excellent profile and was selected for clinical trials. Investigation of N-acyl 4-(5-pyrimidine-2,4-dionyl)phenylalanine derivatives led to the discovery of several very potent dual-acting α4-integrin antagonists. Ethyl ester prodrug 42 advanced to human clinical trials based on the excellent intestinal fluid stability, good permeability and superior efficacy in non-human primates.
Research Interests:
Research Interests:
There is developing evidence that certain thymosin peptides and lymphokines produce a transient increase in steroid hormones when introduced systemically. Conversely, the repressive effect of adrenocortical steroids on the immune system... more
There is developing evidence that certain thymosin peptides and lymphokines produce a transient increase in steroid hormones when introduced systemically. Conversely, the repressive effect of adrenocortical steroids on the immune system is well documented. In the present study, the direct effect of certain components of the immune system on steroid output by rat adrenal fasciculata cells was tested. With this system, there was no direct steroidogenic effect of either the partially purified thymosin fraction 5, or any of the purified peptide components tested (thymosin alpha 1, alpha 7, or beta 4). These peptides also did not synergize the cellular response to ACTH, nor did they induce cAMP production by a ACTH- and NaF-responsive adrenal membrane preparation. Supernatants from Con A-stimulated spleen cells, which were demonstrated to contain lymphokine activity, and partially purified mouse interferon were also without a significant direct or synergistic effect on steroidogenesis by...
Research Interests:
Leukotrienes are vasoactive arachidonic acid metabolites which are released by mast cells during hypersensitivity reactions. The mechanisms for regulating leukotriene biosynthesis are not well understood. A murine mast/basophil cell line... more
Leukotrienes are vasoactive arachidonic acid metabolites which are released by mast cells during hypersensitivity reactions. The mechanisms for regulating leukotriene biosynthesis are not well understood. A murine mast/basophil cell line (PT-18) was used to investigate this problem. Exogenously supplied [14C]arachidonic acid is not appreciably converted to leukotrienes by untreated PT-18 cells. However, when the cells were preincubated with the lymphocyte product 15-hydroxyeicosatetraenoic acid (15-HETE), addition of [14C]arachidonic acid consistently resulted in a dose-dependent synthesis of large amounts of both [14C]leukotriene B4 and [14C]5-HETE. These metabolites were isolated by high pressure liquid chromatography, converted to the methyl ester trimethylsilyl ether derivatives, and the structures confirmed by gas chromatography-mass spectrometry. These findings indicate that 15-HETE induces a direct activation of a cryptic 5-lipoxygenase in these cells. The closely related 12-HETE was ineffective. The activation phenomenon occurs rapidly and is reversible. Furthermore, the activation appears to be highly cell- and enzyme-specific, since lipoxygenases in three primary cell types including one that contains a 5-lipoxygenase and six other cell lines did not show this specific induction of leukotriene biosynthesis by 15-HETE. This report is the first evidence that 15-HETE, a major arachidonate metabolite in lymphocytes, can act as a signal to activate leukotriene production by susceptible mast cells.
Research Interests: Chemistry, Biology, Mast Cells, Biological Chemistry, Medicine, and 14 moreBiological Sciences, Tissue culture, Mice, Animals, Biological, High Pressure Liquid Chromatography, CHEMICAL SCIENCES, Biosynthesis, Arachidonic Acid, Basophil, basophils, Strains, Medical and Health Sciences, and leukotriene
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Research Interests:
Research Interests:
Human rIL-1 alpha was shown to be a potent stimulus of granulopoiesis in mice that have been myelosuppressed with cyclophosphamide. Stimulation of granulopoiesis was demonstrated in IL-1-treated mice by an accelerated recovery of... more
Human rIL-1 alpha was shown to be a potent stimulus of granulopoiesis in mice that have been myelosuppressed with cyclophosphamide. Stimulation of granulopoiesis was demonstrated in IL-1-treated mice by an accelerated recovery of granulocyte-macrophage colony-forming cells, bone marrow and splenic granulocytic hyperplasia, and a profound granulocytosis. Granulopoiesis was stimulated by IL-1 in a dose-dependent manner at doses ranging from 0.5 to 50 micrograms/kg. Maximal increases in granulocytes were observed after 4 days of IL-1 treatment. Mice treated with IL-1 also exhibited increased splenic megakaryopoiesis with a resultant increase in the number of peripheral blood platelets. In contrast to these positive effects on hemopoiesis, bone marrow, but not splenic, erythropoiesis was depressed in IL-1-treated mice. IL-1 effects were observed in mice treated with a wide dose range (50 to 300 mg/kg) of cyclophosphamide, with optimal effects occurring at a dose of 200 mg/kg. The doses ...
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Research Interests:
Research Interests:
The polymerase chain reaction technique (PCR), a primer-mediated enzymatic amplification of specific target sequences, was used to monitor gene transfer into hematopoietic progenitor stem cells. A gene coding for human interleukin 1 alpha... more
The polymerase chain reaction technique (PCR), a primer-mediated enzymatic amplification of specific target sequences, was used to monitor gene transfer into hematopoietic progenitor stem cells. A gene coding for human interleukin 1 alpha (IL-1 alpha) was cotransfected with the rous sarcoma virus (RSV) CAT plasmid into mouse bone marrow cells by electroporation. Individual chloramphenicol (CAM)-resistant bone marrow progenitor colonies (granulocyte-macrophage colony-forming units; CFU-GM) containing 50-100 cells were analyzed by PCR for the presence and expression of IL-1 alpha and CAT sequences. Amplified IL-1 alpha DNA sequences were detected from a 50-cell CFU-GM colony. CAT and IL-1 alpha RNA expression was demonstrated from the CAM-resistant CFU-GM colonies.
Research Interests:
Research Interests: Stem Cells, Biology, Pharmacokinetics, Medicine, Biological Sciences, and 15 moreHumans, Mice, Female, Animals, Male, STEM, Neutrophils, In Vivo, Polyethylene Glycol, Macaca fascicularis, Molecular weight, Antineoplastic Agents, Polyethylene Glycols, High Molecular Weight, and Medical and Health Sciences
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Research Interests:
Research Interests:
Research Interests: Immune response, Immunopharmacology, Spleen, Mice, PWM, and 10 moreAnimals, Male, RIA, Rats, TDT, Body Weight, Tdr, Adrenalectomy, Immunosuppressive Agents, and Thymus Gland
Interleukin-1 (IL-1) is radioprotective and induces both circulating colony-stimulating factor(s) (CSF) and IL-6 in mice. We evaluated the relationship among these three responses to IL-1 using anti-IL-1 receptor antibody 35F5. This... more
Interleukin-1 (IL-1) is radioprotective and induces both circulating colony-stimulating factor(s) (CSF) and IL-6 in mice. We evaluated the relationship among these three responses to IL-1 using anti-IL-1 receptor antibody 35F5. This antibody in vitro blocks responses of T cells and fibroblasts, but not of B cells or myeloid cell lines, to IL-1. Administration of 35F5 alone before irradiation reduced the number of surviving mice compared with those not treated with 35F5, demonstrating that endogenous IL-1 participates in the natural resistance to radiation. Thirty micrograms of 35F5 per mouse also reduced by 92% the survival of irradiated mice pretreated with 0.3 micrograms of IL-1. Similarly, 30 micrograms of 35F5 reduced by 96% to 98% the induction of IL-6 by IL-1. In contrast, 30 micrograms of 35F5 resulted in only moderate reduction of circulating CSF. Consequently, the level of circulating CSF after 35F5 treatment was still equivalent to levels of CSF that were induced by doses ...
Research Interests: Radiation Protection, Cell line, Mice, Animals, Blood, and 15 moreMale, Connective tissue, Monoclonal Antibodies, Bone marrow, Fibroblast Growth Factor, Clinical Sciences, T lymphocytes, Dose Response Relationship, Monoclonal Antibody, Blood cells, Bone Marrow Cells, Interleukin, fibroblasts, Cardiovascular medicine and haematology, and Paediatrics and reproductive medicine
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Research Interests:
Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role... more
Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma.
Research Interests: Lung Inflammation, Dogs, Primates, Humans, Mice, and 15 moreFemale, Animals, Pneumonia, Male, Acute respiratory distress syndrome, Acute Lung Injury, Lung, Lipopolysaccharides, Rats, Ozone, Guinea Pig, Chronic obstructive pulmonary disease, Hypersensitivity, Guinea pigs, and Medical biochemistry and metabolomics
The inhibition of LTB(4) binding to and activation of G-protein-coupled receptors BLT1 and BLT2 is the premise of a treatment for several inflammatory diseases. In a lead optimization effort starting with the leukotriene B(4) (LTB(4))... more
The inhibition of LTB(4) binding to and activation of G-protein-coupled receptors BLT1 and BLT2 is the premise of a treatment for several inflammatory diseases. In a lead optimization effort starting with the leukotriene B(4) (LTB(4)) receptor antagonist (2), members of a series of 3,5-diarylphenyl ethers were found to be highly potent inhibitors of LTB(4) binding to BLT1 and BLT2 receptors, with varying levels of selectivity depending on the substitution. In addition, compounds 33 and 38 from this series have good in vitro ADME properties, good oral bioavailability, and efficacy after oral delivery in guinea pig LTB(4) and nonhuman primate allergen challenge models. Further profiling in a rat non-GLP toxicity experiment provided the rationale for differentiation and selection of one compound (33) for clinical development.
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Although IL-12 has been reported to synergize with c-kit ligand (KL) in promoting hematopoietic stem cell proliferation in vitro, administration of recombinant mouse IL-12 (rIL-12) to normal mice caused a dose- and time-dependent anemia,... more
Although IL-12 has been reported to synergize with c-kit ligand (KL) in promoting hematopoietic stem cell proliferation in vitro, administration of recombinant mouse IL-12 (rIL-12) to normal mice caused a dose- and time-dependent anemia, leukopenia, and thrombocytopenia in vivo. Decreased numbers of bone marrow cells were recovered from the tibiae of IL-12-treated mice, and histologic examination of the marrow revealed a loss of mature neutrophils and red blood cell precursors. However, simultaneously with the suppression of hematopoiesis in the bone marrow, the IL-12-treated mice developed splenomegaly, which was largely caused by a marked enhancement of splenic extramedullary hematopoiesis of the erythroid, myeloid, and megakaryocytic lineages. These histologic observations were confirmed by colony-forming cell assays in which administration of IL-12 was shown to cause a time-dependent decrease in bone marrow CFU-GM, CFU-E, and BFU-E hematopoietic colony-forming cells while causing an increase in splenic CFU-GM and BFU-E colony-forming cells. All these effects were reversible upon cessation of IL-12 treatment. The observation that in IL-12-treated mice hematopoiesis was suppressed in the marrow but enhanced in the spleen suggests that myelosuppression was not caused by a direct effect of IL-12 on hematopoietic progenitors. It seems likely that myelosuppression was caused instead by an IL-12-induced alteration in the local environment of the marrow.
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Research Interests:
Research Interests: Cytokines, Signal Transduction, Cell line, Humans, St, and 15 moreAdaptive Immunity, shRNA, Clinical Sciences, IL, Interleukins, MEF, Chemokines, MAP Kinase, Protein Binding, Biochemistry and cell biology, Chronic myelogenous leukemia, Functional Response, Inflammatory response, Signaling pathway, and Peripheral blood
N-Acyl 4-(5-pyrimidine-2,4-dionyl)phenylalanine derivatives of type 4 were designed to replace the 2,6-dichlorobenzoylamine portion of compound 1 in order to identify novel compounds with improved potency against α4-integrins. Several... more
N-Acyl 4-(5-pyrimidine-2,4-dionyl)phenylalanine derivatives of type 4 were designed to replace the 2,6-dichlorobenzoylamine portion of compound 1 in order to identify novel compounds with improved potency against α4-integrins. Several derivatives were identified as very potent dual-acting α4-integrin, α4β1 and α4β7 antagonists. Investigation of a limited number of prodrug esters led to the discovery of the ethyl ester prodrug 42, which demonstrated good intestinal fluid stability and good permeability. Despite low solubility, 42 gave acceptable blood levels of 30 when dosed orally in non-human primates. Additionally, 42 had an overall excellent profile and was selected for clinical trials. Investigation of N-acyl 4-(5-pyrimidine-2,4-dionyl)phenylalanine derivatives led to the discovery of several very potent dual-acting α4-integrin antagonists. Ethyl ester prodrug 42 advanced to human clinical trials based on the excellent intestinal fluid stability, good permeability and superior efficacy in non-human primates.
Research Interests:
Research Interests:
CRTH2 is one of the prostaglandin D₂ receptors and plays a proinflammatory role in allergic diseases. Gene expression markers in whole blood induced by CRTH2 activation have not previously been reported. Using microarray analyses of 54... more
CRTH2 is one of the prostaglandin D₂ receptors and plays a proinflammatory role in allergic diseases. Gene expression markers in whole blood induced by CRTH2 activation have not previously been reported. Using microarray analyses of 54 675 genes, we revealed modest gene expression changes in human whole blood stimulated in vitro by a selective CRTH2 agonist, DK-PGD₂. Five genes were found to exhibit 1.5- to 2.6-fold changes in expression. The expression of Charcot-Leyden crystal protein/galectin-10 (CLC/Gal-10) in particular was consistently enhanced in human whole blood stimulated by DK-PGD₂, as confirmed by quantitative real-time polymerase chain reaction analyses. DK-PGD(2)-induced increases in blood CLC/Gal-10 mRNA levels were largely attenuated by the CRTH2 antagonist CAY10471.Thus, the DK-PGD₂-induced CLC/Gal-10 mRNA level can serve as a potential marker for monitoring pharmacodynamic effects of blood exposure to CRTH2 modulating agents.