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1.
Pediatr Obes ; 13(1): 70-73, 2018 01.
Article in English | MEDLINE | ID: mdl-27780309

ABSTRACT

Hepatocellular adenomas (HCA) are benign tumours with potential for malignant transformation with no recommendations regarding management in the paediatric population. We report a case of an inflammatory adenoma with ß-catenin activated pathway in an obese, paediatric patient with nonalcoholic steatohepatitis (NASH). CASE REPORT: An 11-year-old female presented with a microlobulated liver lesion measuring >5 cm in magnetic resonance imaging (MRI) with inflammatory adenoma with ß-catenin activated pathology arising in a background of NASH, nonalcoholic fatty liver disease (NAFLD) activity score 5/8. Imaging follow-up demonstrated stable disease without progression for 3 years. DISCUSSION: Malignant transformation of Hepatocellular adenomas in a child is approximately 4.2%. It is unknown if hepatic steatosis increases this risk. Obese patients mainly develop inflammatory and ß-catenin activated (highest risk for malignant transformation) adenomas. Our patient had inflammatory and ß-catenin activation, which led to monitoring for malignant transformation. CONCLUSION: We report a ß-catenin activated inflammatory adenoma in a child with obesity and NASH with ongoing expectant management.


Subject(s)
Adenoma, Liver Cell/complications , Liver Neoplasms/pathology , Non-alcoholic Fatty Liver Disease/complications , Pediatric Obesity/complications , beta Catenin/metabolism , Adenoma, Liver Cell/metabolism , Cell Transformation, Neoplastic , Child , Female , Humans , Liver Neoplasms/metabolism , Magnetic Resonance Imaging
2.
Transfusion ; 57(11): 2690-2700, 2017 11.
Article in English | MEDLINE | ID: mdl-28880373

ABSTRACT

BACKGROUND: Although transfusion is a lifesaving intervention, it may be associated with significant morbidity in injured patients. We hypothesize that stored red blood cells (RBCs) induce proinflammatory activation of human pulmonary microvascular endothelial cells (HMVECs) resulting in neutrophil (PMN) adhesion and predisposition to acute lung injury (ALI). STUDY DESIGN AND METHODS: Ten units of RBCs were collected; 50% (by weight) were leukoreduced (LR-RBCs) and the remainder was unmodified and stored in additive solution-5 (AS-5). An additional 10 units of RBCs were collected, leukoreduced, and stored in AS-3. HMVECs were incubated with [10%-40%]FINAL of the supernatants on Day (D)1 to D42 of storage, lipid extracts, and purified lipids. Endothelial surface expression of intercellular adhesion molecule-1 (ICAM-1), interleukin (IL)-8 release, and PMN adhesion to HMVECs were measured. HMVEC signaling via the BLT2 receptor was evaluated. Supernatants and lipids were also employed as the first event in a two-event model of ALI. RESULTS: The supernatants [10%-40%]FINAL from D21 LR-RBCs and D42 RBCs and LR-RBCs and the lipids from D42 stored in AS-5 induced increased ICAM-1 surface expression on endothelium, IL-8 release, and PMN adhesion. In addition, the supernatants [20%-40%]FINAL from D21 and D42 RBCs in AS-5 also increased endothelial surface expression of ICAM-1. D42 supernatants and lipids also caused coprecipitation of ß-arrestin-1 with BLT2, protein kinase C (PKC)ßI , and PKCδ and served as the first event in a two-event rodent model of ALI. CONCLUSION: Lipids that accumulate during RBC storage activate endothelium and predispose to ALI, which may explain some of the adverse events associated with the transfusion of critically injured patients.


Subject(s)
Blood Preservation/methods , Erythrocytes/cytology , Lipids/pharmacology , Lung/blood supply , Protein Kinase C/metabolism , Receptors, Leukotriene B4/metabolism , Acute Lung Injury/etiology , Culture Media, Conditioned/pharmacology , Endothelial Cells/metabolism , Enzyme Activation , Erythrocyte Transfusion/adverse effects , Humans , Microvessels/cytology , Pneumonia/etiology
3.
J Leukoc Biol ; 101(1): 261-273, 2017 01.
Article in English | MEDLINE | ID: mdl-27531930

ABSTRACT

Lysophosphatidylcholines (lysoPCs) are effective polymorphonuclear neutrophil (PMN) priming agents implicated in transfusion-related acute lung injury (TRALI). LysoPCs cause ligation of the G2A receptor, cytosolic Ca2+ flux, and activation of Hck. We hypothesize that lysoPCs induce Hck-dependent activation of protein kinase C (PKC), resulting in phosphorylation and membrane translocation of 47 kDa phagocyte oxidase protein (p47phox). PMNs, human or murine, were primed with lysoPCs and were smeared onto slides and examined by digital microscopy or separated into subcellular fractions or whole-cell lysates. Proteins were immunoprecipitated or separated by polyacrylamide gel electrophoresis and immunoblotted for proteins of interest. Wild-type (WT) and PKCγ knockout (KO) mice were used in a 2-event model of TRALI. LysoPCs induced Hck coprecipitation with PKCδ and PKCγ and the PKCδ:PKCγ complex also had a fluorescence resonance energy transfer (FRET)+ interaction with lipid rafts and Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2 (WAVE2). PKCγ then coprecipitated with p47phox Immunoblotting, immunoprecipitation (IP), specific inhibitors, intracellular depletion of PKC isoforms, and PMNs from PKCγ KO mice demonstrated that Hck elicited activation/Tyr phosphorylation (Tyr311 and Tyr525) of PKCδ, which became Thr phosphorylated (Thr507). Activated PKCδ then caused activation of PKCγ, both by Tyr phosphorylation (Τyr514) and Ser phosphorylation, which induced phosphorylation and membrane translocation of p47phox In PKCγ KO PMNs, lysoPCs induced Hck translocation but did not evidence a FRET+ interaction between PKCδ and PKCγ nor prime PMNs. In WT mice, lysoPCs served as the second event in a 2-event in vivo model of TRALI but did not induce TRALI in PKCγ KO mice. We conclude that lysoPCs prime PMNs through Hck-dependent activation of PKCδ, which stimulates PKCγ, resulting in translocation of phosphorylated p47phox.


Subject(s)
Cell Membrane/metabolism , Lysophosphatidylcholines/pharmacology , NADPH Oxidases/metabolism , Neutrophils/metabolism , Protein Kinase C-delta/metabolism , Protein Kinase C/metabolism , Proto-Oncogene Proteins c-hck/metabolism , Animals , Calcium/metabolism , Cell Membrane/drug effects , Enzyme Activation/drug effects , Humans , Lung Injury/pathology , Mice , Mice, Knockout , Neutrophils/drug effects , Phosphorylation/drug effects , Protein Binding/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Transport/drug effects , Recombinant Proteins/pharmacology
4.
Pediatr Blood Cancer ; 58(3): 399-405, 2012 Mar.
Article in English | MEDLINE | ID: mdl-21793188

ABSTRACT

BACKGROUND: Acute chest syndrome (ACS) in sickle cell disease is associated with elevation of secretory phospholipase A(2) (sPLA(2) ). We hypothesize that sPLA(2) cleaves membrane lipids from sickled red blood cells (RBCs) causing PMN-mediated endothelial cell injury (ECI) as the second event in a two-event model. METHODS: Whole blood was collected from children when in steady state or daily during admissions for vaso-occlusive pain (VOC) or ACS. The plasma and RBCs were separated, sPLA(2) levels were measured, and the RBCs were incubated with sPLA(2) . Plasma and lipids, extracted from the plasma or the supernatant of sPLA(2) -treated RBCs, were assayed for PMN priming activity and used as the second event in a model of PMN-mediated ECI. Phosphatidylserine (PS) surface expression on RBCs was quantified by flow cytometry. RESULTS: Increased sPLA(2) -IIa levels were associated with ACS. SPLA(2) -liberated lipids from VOC and the plasma, plasma lipids and sPLA(2) -liberated lipids from ACS primed PMNs and caused PMN-mediated ECI (P < 0.01). RBCs from VOC had increased in PS surface expression versus steady state. CONCLUSIONS: ACS plasma and lipids and sPLA(2) -released lipids from RBCs during VOC or ACS induce PMN-mediated ECI. VOC elicited increases in PS surface expression providing a membrane substrate for sPLA(2) lysis of sickle RBCs.


Subject(s)
Acute Chest Syndrome/physiopathology , Neutrophils/metabolism , Phospholipases A2, Secretory/blood , Adolescent , Child , Child, Preschool , Colorado , Endothelium, Vascular , Female , Humans , Infant , Lung/blood supply , Male
5.
Shock ; 35(3): 240-4, 2011 Mar.
Article in English | MEDLINE | ID: mdl-20926984

ABSTRACT

Leukotrienes are proinflammatory lipid mediators, derived from arachidonic acid via 5-lipoxygenase (5-LO). Leukotriene B4 (LTB4) is an effective polymorphonuclear neutrophil (PMN) chemoattractant, as well as being a major product of PMN priming. Leukotriene B4 is rapidly metabolized into products that are thought to be inactive, and little is known about the effects of LTB4 on the pulmonary endothelium. We hypothesize that LTB4 and its metabolites are effective PMN priming agents and cause proinflammatory activation of pulmonary endothelial cells. Isolated PMNs were primed (5 min, 37°C) with serial concentrations 10 to 10 M of LTB4 and its metabolites: 6-trans-LTB4, 20-OH-LTB4, and 20-COOH-LTB4, and then activated with fMLP. Primary human pulmonary microvascular endothelial cells (HMVECs) were incubated with these lipids (6 h, 37°C, 5% CO2), and intercellular adhesion molecule 1 was measured by flow cytometry. Polymorphonuclear neutrophil adhesion was measured by myeloperoxidase assays, and to ensure that these reactions were specific to the LTB4 receptors, BLT1 and BLT2 were antagonized with CP105,696 (BLT1) or silenced with siRNA (BLT1 and BLT2). Leukotriene B4 and its metabolites primed PMNs over a wide range of concentrations, depending on the specific metabolite. In addition, at high concentrations these lipids also caused increases in the surface expression of intercellular adhesion molecule 1 on HMVECs and induced HMVEC-mediated adhesion of PMNs. Silencing of BLT2 abrogated HMVEC activation, and blockade of BLT1 inhibited the observed PMN priming activity. We conclude that LTB4 and its ω-oxidation and nonenzymatic metabolites prime PMNs over a range of concentrations and activate HMVECs. These data have expanded the repertoire of causative agents in acute lung injury and postinjury multiple organ failure.


Subject(s)
Endothelial Cells/metabolism , Neutrophils/enzymology , Neutrophils/metabolism , Oxidoreductases/metabolism , Cells, Cultured , Chromatography, High Pressure Liquid , Endothelial Cells/immunology , Humans , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Leukotriene B4/genetics , Leukotriene B4/metabolism , Lung/cytology , Lung/metabolism , Neutrophils/immunology , RNA Interference , Receptors, Leukotriene B4/genetics , Receptors, Leukotriene B4/metabolism
6.
Biochem J ; 432(1): 35-45, 2010 Nov 15.
Article in English | MEDLINE | ID: mdl-20799926

ABSTRACT

Lyso-PCs (lysophosphatidylcholines) are a mixture of lipids that accumulate during storage of cellular blood components, have been implicated in TRALI (transfusion-related acute lung injury) and directly affect the physiology of neutrophils [PMNs (polymorphonuclear leucocytes)]. Because the G2A receptor, expressed on PMNs, has been reported to recognize lyso-PCs, we hypothesize that lyso-PC activation of G2A causes the increases in cytosolic Ca²(+) via release of G(α) and G(ßγ) subunits, kinase activation, and the recruitment of clathrin, ß-arrestin-1 and GRK6 (G-protein receptor kinase 6) to G2A for signal transduction. PMNs were isolated by standard techniques, primed with lyso-PCs for 5-180 s, and lysed for Western blot analysis, immunoprecipitation or subcellular fractionation, or fixed and smeared on to slides for digital microscopy. The results demonstrated that lyso-PCs cause rapid activation of the G2A receptor through S-phosphorylation and internalization resulting in G(αi)₋1 and G(αq/)11 release leading to increases in cytosolic Ca²(+), which was inhibited by an antibody to G2A or intracellular neutralization of these subunits. Lyso-PCs also caused the release of the G(ßγ) subunit which demonstrated a physical interaction (FRET+) with activated Hck (haemopoietic cell kinase; Tyr4¹¹). Moreover, G2A recruited clathrin, ß-arrestin-1 and GRK6: clathrin is important for signal transduction, GRK6 for receptor de-sensitization, and ß-arrestin-1 both propagates and terminates signals. We conclude that lyso-PC activation of G2A caused release of G(αi)₋1, G(αq/)11 and G(ßγ), resulting in cytosolic Ca²(+) flux, Hck activation, and recruitment of clathrin, ß-arrestin-1 and GRK6.


Subject(s)
Calcium/metabolism , Cell Cycle Proteins/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Lysophosphatidylcholines/pharmacology , Neutrophils/drug effects , Protein Kinases/metabolism , Receptors, G-Protein-Coupled/metabolism , Arrestins/metabolism , Blotting, Western , Cells, Cultured , Clathrin/metabolism , Cytosol/drug effects , Cytosol/metabolism , Enzyme Activation/drug effects , Fluorescence Resonance Energy Transfer , G-Protein-Coupled Receptor Kinases/metabolism , GTP-Binding Protein alpha Subunits/metabolism , GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/metabolism , Humans , Ion Transport/drug effects , Microscopy, Fluorescence/methods , Neutrophils/cytology , Neutrophils/metabolism , Phosphorylation/drug effects , Proto-Oncogene Proteins c-hck/metabolism , Signal Transduction/drug effects , Time Factors , beta-Arrestin 1 , beta-Arrestins
7.
Am J Physiol Cell Physiol ; 298(3): C714-24, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19907017

ABSTRACT

Neutrophils (PMNs) are a vital part of host defense and are the principal leukocyte in innate immunity. Interleukin (IL)-18 is a proinflammatory cytokine with roles in both innate and adaptive immunity. We hypothesize that PMNs contain preformed IL-18, which is released in response to specific inflammatory stimuli. Isolated PMNs were stimulated with a battery of chemoattractants (5 min to 24 h), and IL-18 release was measured. PMNs were also separated into subcellular fractions and immunoblotted with antibodies against IL-18 or were fixed and probed with antibodies to IL-18 as well as to the contents of granules, intracellular organelles, and filamentous actin (F-actin), incubated with fluorescent secondary antibodies, and examined by digital microscopy. Quiescent PMNs contained IL-18 in the cytoplasm, associated with F-actin, as determined by positive fluorescence resonance energy transfer (FRET+). In turn, TNF-alpha stimulation disrupted the association of IL-18 with F-actin, induced a FRET+ interaction of IL-18 with lipid rafts, and elicited IL-18 release. Manipulation of F-actin status confirmed the relationship between IL-18 and F-actin in resting PMNs. Consequently, incubation with monomeric IL-18 binding protein inhibited TNF-alpha-mediated priming of the PMN oxidase. We conclude that human PMNs contain IL-18 associated with F-actin in the cytoplasm and TNF-alpha stimulation causes dissociation of IL-18 from F-actin, association with lipid rafts, and extracellular release. Extracellular IL-18 participates in TNF-alpha priming of the PMN oxidase as demonstrated by inhibition with the IL-18 binding protein.


Subject(s)
Cytosol/immunology , Inflammation Mediators/metabolism , Interleukin-18/metabolism , Neutrophils/immunology , Tumor Necrosis Factor-alpha/metabolism , Actins/metabolism , Adaptive Immunity , Fluorescence Resonance Energy Transfer , Fluorescent Antibody Technique, Indirect , Humans , Immunity, Innate , Immunoblotting , Intercellular Signaling Peptides and Proteins/metabolism , Membrane Microdomains/immunology , Microscopy, Fluorescence , Phosphoproteins/metabolism , Time Factors
8.
Am J Physiol Cell Physiol ; 297(4): C886-97, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19295175

ABSTRACT

Receptor signaling is integral for adhesion, emigration, phagocytosis, and reactive oxygen species production in polymorphonuclear neutrophils (PMNs). Priming is an important part of PMN emigration, but it can also lead to PMN-mediated organ injury in the host. Platelet-activating factor (PAF) primes PMNs through activation of a specific G protein-coupled receptor. We hypothesize that PAF priming of PMNs requires clathrin-mediated endocytosis (CME) of the PAF receptor (PAFr), and, therefore, amantadine, known to inhibit CME, significantly antagonizes PAF signaling. PMNs were isolated by standard techniques to >98% purity and tested for viability. Amantadine (1 mM) significantly inhibited the PAF-mediated changes in the cellular distribution of clathrin and the physical colocalization [fluorescence resonance energy transfer positive (FRET+)] of early endosome antigen-1 and Rab5a, known components of CME and similar to hypertonic saline, a known inhibitor of CME. Furthermore, amantadine had no effect on the PAF-induced cytosolic calcium flux; however, phosphorylation of p38 MAPK was significantly decreased. Amantadine inhibited PAF-mediated changes in PMN physiology, including priming of the NADPH oxidase and shape change with lesser inhibition of increases in CD11b surface expression and elastase release. Furthermore, rimantadine, an amantadine analog, was a more potent inhibitor of PAF priming of the N-formyl-methionyl-leucyl-phenylalanine-activated oxidase. PAF priming of PMNs requires clathrin-mediated endocytosis that is inhibited when PMNs are pretreated with either amantadine or rimantadine. Thus, amantadine and rimantadine have the potential to ameliorate PMN-mediated tissue damage in humans.


Subject(s)
Amantadine/pharmacology , Clathrin/metabolism , Endocytosis , Neutrophils/physiology , Platelet Activating Factor/physiology , Platelet Membrane Glycoproteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Antigens, CD1/metabolism , Enzyme Activation , Humans , In Vitro Techniques , NADPH Oxidases/metabolism , Neutrophils/drug effects , Platelet Activating Factor/pharmacology , Platelet Membrane Glycoproteins/antagonists & inhibitors , Receptors, G-Protein-Coupled/antagonists & inhibitors , Rimantadine/pharmacology , Signal Transduction
9.
J Immunol ; 180(12): 8192-203, 2008 Jun 15.
Article in English | MEDLINE | ID: mdl-18523285

ABSTRACT

Neutrophils (polymorphonuclear leukocytes, PMNs) are vital to innate immunity and receive proinflammatory signals that activate G protein-coupled receptors (GPCRs). Because GPCRs transduce signals through clathrin-mediated endocytosis (CME), we hypothesized that platelet-activating factor (PAF), an effective chemoattractant that primes the PMN oxidase, would signal through CME, specifically via dynamin-2 activation and endosomal formation resulting in membrane translocation of cytosolic phagocyte oxidase (phox) proteins. PMNs were incubated with buffer or 2 muM PAF for 1-3 min, and in some cases activated with PMA, and O(2)(-) was measured, whole-cell lysates and subcellular fractions were prepared, or the PMNs were fixed onto slides for digital or electron microscopy. PAF caused activation of dynamin-2, resulting in endosomal formation that required PI3K and contained early endosomal Ag-1 (EEA-1) and Rab5a. The apoptosis signal-regulating kinase-1/MAPK kinase-3/p38 MAPK signalosome assembled on Rab5a and phosphorylated EEA-1 and Rab GDP dissociation inhibitor, with the latter causing Rab5a activation. Electron microscopy demonstrated that PAF caused two distinct sites for activation of p38 MAPK. EEA-1 provided a scaffold for recruitment of the p40(phox)-p67(phox) complex and PI3K-dependent Akt1 phosphorylation of these two phox proteins. PAF induced membrane translocation of p40(phox)-p67(phox) localizing to gp91(phox), which was PI3K-, but not p47(phox)-, dependent. In conclusion, PAF transduces signals through CME, and such GPCR signaling may allow for pharmacological manipulation of these cells to decrease PMN-mediated acute organ injury.


Subject(s)
Cell Membrane/metabolism , Endosomes/metabolism , Neutrophils/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/metabolism , Platelet Activating Factor/physiology , p38 Mitogen-Activated Protein Kinases/metabolism , rab5 GTP-Binding Proteins/metabolism , Cell Membrane/enzymology , Dynamin II/metabolism , Endosomes/enzymology , Enzyme Activation/physiology , Fluorescence Resonance Energy Transfer , Humans , Ligands , MAP Kinase Signaling System/physiology , Neutrophils/enzymology , Platelet Activating Factor/metabolism , Platelet Membrane Glycoproteins/metabolism , Protein Transport/physiology , Receptors, G-Protein-Coupled/metabolism , Vesicular Transport Proteins/metabolism , p38 Mitogen-Activated Protein Kinases/physiology
10.
J Immunol ; 176(11): 7039-50, 2006 Jun 01.
Article in English | MEDLINE | ID: mdl-16709866

ABSTRACT

Clathrin-mediated endocytosis (CME) is a common pathway used by G protein-linked receptors to transduce extracellular signals. We hypothesize that platelet-activating factor (PAF) receptor (PAFR) ligation requires CME and causes engagement of beta-arrestin-1 and recruitment of a p38 MAPK signalosome that elicits distinct actin rearrangement at the receptor before endosomal scission. Polymorphonuclear neutrophils were stimulated with buffer or 2 microM PAF (1 min), and whole cell lysates or subcellular fractions were immunoprecipitated or slides prepared for colocalization and fluorescent resonance energy transfer analysis. In select experiments, beta-arrestin-1 or dynamin-2 were neutralized by intracellular introduction of specific Abs. PAFR ligation caused 1) coprecipitation of the PAFR and clathrin with beta-arrestin-1, 2) fluorescent resonance energy transfer-positive interactions among the PAFR, beta-arrestin-1, and clathrin, 3) recruitment and activation of the apoptosis signal-regulating kinase-1/MAPK kinase-3/p38 MAPK (ASK1/MKK3/p38 MAPK) signalosome, 4) cell polarization, and 5) distinct actin bundle formation at the PAFR. Neutralization of beta-arrestin-1 inhibited all of these cellular events, including PAFR internalization; conversely, dynamin-2 inhibition only affected receptor internalization. Selective p38 MAPK inhibition globally abrogated actin rearrangement; however, inhibition of MAPK-activated protein kinase-2 and its downstream kinase leukocyte-specific protein-1 inhibited only actin bundle formation and PAFR internalization. In addition, ASK1/MKK3/p38 MAPK signalosome assembly appears to occur in a novel manner such that the ASK1/p38 MAPK heterodimer is recruited to a beta-arrestin-1 bound MKK3. In polymorphonuclear neutrophils, leukocyte-specific protein-1 may play a role similar to fascin for actin bundle formation. We conclude that PAF signaling requires CME, beta-arrestin-1 recruitment of a p38 MAPK signalosome, and specific actin bundle formation at the PAFR for transduction before endosomal scission.


Subject(s)
Actins/metabolism , Arrestins/metabolism , Cell Membrane/enzymology , Clathrin/physiology , Endocytosis/physiology , MAP Kinase Signaling System/physiology , Platelet Activating Factor/physiology , p38 Mitogen-Activated Protein Kinases/physiology , Arrestins/physiology , Calcium/metabolism , Cell Membrane/metabolism , Clathrin/metabolism , Dynamin II/physiology , Endosomes/enzymology , Endosomes/metabolism , Enzyme Activation/physiology , Humans , MAP Kinase Kinase Kinase 5/isolation & purification , MAP Kinase Kinase Kinase 5/metabolism , Microfilament Proteins/metabolism , Microfilament Proteins/physiology , Neutrophils/cytology , Neutrophils/enzymology , Neutrophils/metabolism , Platelet Activating Factor/metabolism , Platelet Membrane Glycoproteins/metabolism , Platelet Membrane Glycoproteins/physiology , Protein Transport/physiology , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/physiology , Subcellular Fractions/enzymology , beta-Arrestin 1 , beta-Arrestins , p38 Mitogen-Activated Protein Kinases/metabolism
11.
Blood Rev ; 20(3): 139-59, 2006 May.
Article in English | MEDLINE | ID: mdl-16360246

ABSTRACT

Transfusion-related acute lung injury (TRALI) is a life-threatening adverse event of transfusion, which has an increasing incidence in the United States and is the leading cause of transfusion-related death. TRALI and acute lung injury (ALI) share a common clinical definition except that TRALI is temporally- and mechanistically-related to transfusion of blood or blood components. A number of different models have been proposed to explain the pathogenesis. The first is an antibody-mediated event whereby transfusion of anti-HLA, class I or class II, or anti-granulocyte antibodies into patients whose leukocytes express the cognate antigens. The antibody:antigen interaction causes complement-mediated pulmonary sequestration and activation of neutrophils (PMNs) resulting in TRALI. The second is a two-event model: the first event is the clinical condition of the patient resulting in pulmonary endothelial activation and PMN sequestration, and the second event is the transfusion of a biologic response modifier (including anti-granulocyte antibodies, lipids, and CD40 ligand) that activates these adherent PMNs resulting in endothelial damage, capillary leak, and TRALI. These hypotheses are discussed with respect to animal models and human studies that provide the experimental and clinical relevance. The definition of TRALI, patient predisposition, treatment, prevention and reporting guidelines are also examined.


Subject(s)
Lung Diseases/etiology , Transfusion Reaction , Animals , Antibodies/immunology , Diagnosis, Differential , Humans , Lung Diseases/diagnosis , Lung Diseases/epidemiology , Lung Diseases/immunology , Lung Diseases/pathology
12.
J Leukoc Biol ; 78(5): 1025-42, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16204621

ABSTRACT

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is part of the microbicidal arsenal used by human polymorphonuclear neutrophils (PMNs) to eradicate invading pathogens. The production of a superoxide anion (O2-) into the phagolysosome is the precursor for the generation of more potent products, such as hydrogen peroxide and hypochlorite. However, this production of O2- is dependent on translocation of the oxidase subunits, including gp91phox, p22phox, p47phox, p67phox, p40phox, and Rac2 from the cytosol or specific granules to the plasma membrane. In response to an external stimuli, PMNs change from a resting, nonadhesive state to a primed, adherent phenotype, which allows for margination from the vasculature into the tissue and chemotaxis to the site of infection upon activation. Depending on the stimuli, primed PMNs display altered structural organization of the NADPH oxidase, in that there is phosphorylation of the oxidase subunits and/or translocation from the cytosol to the plasma or granular membrane, but there is not the complete assembly required for O2- generation. Activation of PMNs is the complete assembly of the membrane-linked and cytosolic NADPH oxidase components on a PMN membrane, the plasma or granular membrane. This review will discuss the individual components associated with the NADPH oxidase complex and the function of each of these units in each physiologic stage of the PMN: rested, primed, and activated.


Subject(s)
NADPH Oxidases/immunology , Neutrophils/enzymology , Protein Subunits/immunology , Cell Membrane/enzymology , Cell Membrane/immunology , Cytosol/enzymology , Humans , Models, Immunological , NADPH Oxidases/chemistry , Neutrophils/immunology , Phosphorylation , Protein Subunits/chemistry , Superoxides/immunology
13.
Am J Physiol Endocrinol Metab ; 288(4): E775-81, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15572655

ABSTRACT

Testosterone (Te) concentrations fall gradually in healthy aging men. Postulated mechanisms include relative failure of gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), and/or gonadal Te secretion. Available methods to test Leydig cell Te production include pharmacological stimulation with human chorionic gonadotropin (hCG). We reasoned that physiological lutropic signaling could be mimicked by pulsatile infusion of recombinant human (rh) LH during acute suppression of LH secretion. To this end, we studied eight young (ages 19-30 yr) and seven older (ages 61-73 yr) men in an experimental paradigm comprising 1) inhibition of overnight LH secretion with a potent selective GnRH-receptor antagonist (ganirelix, 2 mg sc), 2) intravenous infusion of consecutive pulses of rh LH (50 IU every 2 h), and 3) chemiluminometric assay of LH and Te concentrations sampled every 10 min for 26 h. Statistical analyses revealed that 1) ganirelix suppressed LH and Te equally (> 75% median inhibition) in young and older men, 2) infused LH pulse profiles did not differ by age, and 3) successive intravenous pulses of rh LH increased concentrations of free Te (ng/dl) to 4.6 +/- 0.38 (young) and 2.1 +/- 0.14 (older; P < 0.001) and bioavailable Te (ng/dl) to 337 +/- 20 (young) and 209 +/- 16 (older; P = 0.002). Thus controlled pulsatile rh LH drive that emulates physiological LH pulses unmasks significant impairment of short-term Leydig cell steroidogenesis in aging men. Whether more prolonged pulsatile LH stimulation would normalize this inferred defect is unknown.


Subject(s)
Aging/metabolism , Gonadotropin-Releasing Hormone/analogs & derivatives , Luteinizing Hormone/administration & dosage , Receptors, LHRH/antagonists & inhibitors , Testis/drug effects , Testosterone/biosynthesis , Adult , Aged , Biological Availability , Gonadotropin-Releasing Hormone/pharmacology , Hormone Antagonists/pharmacology , Humans , Infusions, Intravenous , Luteinizing Hormone/antagonists & inhibitors , Male , Middle Aged , Testis/metabolism , Testosterone/blood
14.
Am J Physiol Cell Physiol ; 283(5): C1522-9, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12372813

ABSTRACT

COOH-terminal cytoplasmic tails of chloride/bicarbonate anion exchangers (AE) bind cytosolic carbonic anhydrase II (CAII) to form a bicarbonate transport metabolon, a membrane protein complex that accelerates transmembrane bicarbonate flux. To determine whether interaction with CAII affects the downregulated in adenoma (DRA) chloride/bicarbonate exchanger, anion exchange activity of DRA-transfected HEK-293 cells was monitored by following changes in intracellular pH associated with bicarbonate transport. DRA-mediated bicarbonate transport activity of 18 +/- 1 mM H+ equivalents/min was inhibited 53 +/- 2% by 100 mM of the CAII inhibitor, acetazolamide, but was unaffected by the membrane-impermeant carbonic anhydrase inhibitor, 1-[5-sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-dimethyl-4-phenyl-pyridinium perchlorate. Compared with AE1, the COOH-terminal tail of DRA interacted weakly with CAII. Overexpression of a functionally inactive CAII mutant, V143Y, reduced AE1 transport activity by 61 +/- 4% without effect on DRA transport activity (105 +/- 7% transport activity relative to DRA alone). We conclude that cytosolic CAII is required for full DRA-mediated bicarbonate transport. However, DRA differs from other bicarbonate transport proteins because its transport activity is not stimulated by direct interaction with CAII.


Subject(s)
Antiporters , Bicarbonates/metabolism , Carbonic Anhydrase II/metabolism , Carrier Proteins/metabolism , Membrane Proteins/metabolism , Acetazolamide/pharmacology , Adenoma/metabolism , Carbonic Anhydrase II/antagonists & inhibitors , Carbonic Anhydrase Inhibitors/pharmacology , Carrier Proteins/genetics , Cell Line , Cell Membrane/metabolism , Chloride-Bicarbonate Antiporters , Chlorides/metabolism , Down-Regulation , Humans , Membrane Proteins/genetics , Protein Binding/physiology , Sulfate Transporters , Transfection
15.
Am J Physiol Gastrointest Liver Physiol ; 281(5): G1322-8, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11668042

ABSTRACT

We examined whether the capsaicin vanilloid receptor-1 (VR1) mediates substance P (SP) release from primary sensory neurons in experimental pancreatitis. Pancreatitis was achieved by 12 hourly injections of caerulein (50 microg/kg ip) in mice. One group received capsazepine (100 micromol/kg sc), a competitive VR1 antagonist, at 4-h intervals. Neurokinin-1 receptor (NK1R) internalization in acinar cells, used as an index of endogenous SP release, was assessed by immunocytochemical quantification of NK1R endocytosis. The severity of pancreatitis was assessed by measurements of serum amylase, pancreatic myeloperoxidase (MPO) activity, and histological grading. Caerulein administration caused significant elevations in serum amylase and pancreatic MPO activity, produced histological evidence of pancreatitis, and caused a dramatic increase in NK1R endocytosis. Capsazepine treatment significantly reduced the level of NK1R endocytosis, and this was associated with similar reductions in pancreatic MPO activity and histological severity of pancreatitis. These results demonstrate that repeated caerulein stimulation causes experimental pancreatitis that is mediated in part by stimulation of VR1 on primary sensory neurons, resulting in endogenous SP release.


Subject(s)
Capsaicin/analogs & derivatives , Pancreatitis/metabolism , Receptors, Drug/physiology , Substance P/metabolism , Amylases/blood , Animals , Capsaicin/pharmacology , Endocytosis , Male , Mice , Mice, Inbred C57BL , Pancreas/drug effects , Pancreas/enzymology , Pancreas/pathology , Pancreas/physiopathology , Pancreatitis/pathology , Pancreatitis/physiopathology , Peroxidase/metabolism , Receptors, Neurokinin-1/metabolism , Severity of Illness Index , Substance P/antagonists & inhibitors , TRPV Cation Channels
16.
Pancreas ; 22(2): 172-8, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11249072

ABSTRACT

Chronic pancreatitis (CP) is associated with impaired glucose tolerance and with reduced hepatic sensitivity to insulin. We have previously shown that in normal and sham-operated rats, insulin suppresses hepatic glucose production, and this suppression is associated with a decrease in the hepatocyte plasma membrane-bound quantity of the facilitative glucose transport protein GLUT2. The insulin-mediated reduction in membrane-bound GLUT2 is impaired in CP, and may play a role in the glucose intolerance associated with CP. To determine whether GLUT2 is actively internalized and whether this mechanism is disordered in CP, livers from fed and fasting rats in whom CP had been induced 2-3 months earlier by pancreatic duct oleic acid infusion, and in sham-operated (sham) rats, were fractionated to yield endosome (E)- and plasma membrane (PM)-enriched fractions. Forty-five minutes after duodenal intubation alone (fasting) or intubation plus duodenal feeding, livers were removed, homogenized and ultracentrifuged, and microsomal pellets were separated by sucrose density gradient ultracentrifugation. GLUT2 content of fractions was determined by Western blotting and scanning densitometry. The E:PM ratio of GLUT2 increased from 0.68 +/- 0.11 (mean +/- SEM) in fasting sham livers (n = 8) to 1.04 +/- 0.09 in fed sham livers (n = 8; p < 0.05). However, there was no change in the E:PM ratio of GLUT2 in CP livers after duodenal feeding (0.90 +/- 0.12 vs. 0.86 +/- 0.10; n = 8,8; p = NS). To test our findings using confocal laser scanning microscopy, liver specimens from fed and fasting CP and sham rats were minced, fixed in 4% paraformaldehyde, sectioned, and stained with rabbit antirat GLUT2 antibody followed by rhodamine-labeled secondary antibody. GLUT2 was quantified by mean pixel intensity in an 8 x 16-pixel area of PM and a 16 x 16-pixel area of cytosol (CYT) in each of 30 random cells/field (400x) in each of three rats per group. As in the fractionation study, duodenal feeding increased the CYT:PM ratio of GLUT2 from 0.75 +/- 0.01 in fasting sham liver to 0.86 +/- 0.01 in fed sham liver (p < 0.0001), while the CYT:PM ratio in CP remained unchanged. We conclude that feeding induces a shift in GLUT2 from the plasma membrane to the endosomal pool. The feeding-induced internalization of GLUT2 is absent in livers from rats with CP and may play a role in the glucose intolerance associated with CP.


Subject(s)
Hepatocytes/metabolism , Monosaccharide Transport Proteins/metabolism , Pancreatitis/metabolism , Animals , Blotting, Western , Chronic Disease , Glucose Transporter Type 2 , Male , Microscopy, Confocal , Rats , Rats, Sprague-Dawley
17.
Yale J Biol Med ; 74(1): 13-20, 2001.
Article in English | MEDLINE | ID: mdl-11249235

ABSTRACT

BACKGROUND: Intra-abdominal desmoplastic small round cell tumor is a rare malignancy with a predilection for young males. Unique histological and immunocytochemical features distinguish the tumor from other members of the family of small round cell tumors of infancy and childhood. The aggressive nature of tumor spread, relative insensitivity to chemotherapy, and generally incomplete resectability result in a very poor prognosis. The authors report a case of a 39-year-old man with diffuse abdominal and pelvic involvement of intra-abdominal desmoplastic small round cell tumor treated with aggressive chemotherapy and surgery. METHODS: Computed tomography (CT)-guided biopsy of an omental mass was performed. Histologically, discrete nests of uniform closely packed malignant cells were distributed in a background of focally desmoplastic stroma. Immunocytochemistry demonstrated positivity for epithelial, mesenchymal, and neural markers. On the basis of these unique histological and immunohistochemical characteristics, the diagnosis of desmoplastic small round cell tumor was made. The patient was treated with aggressive neoadjuvant chemotherapy consisting of a high-dose alkylator -based combination regimen, followed by surgery. RESULTS: The patient had a 10 to 15 percent regression in tumor mass in response to chemotherapy. Laparotomy revealed two large omental masses, another large mass adherent to the left colon and pelvic sidewall, and diaphragmatic, peritoneal and mesenteric studding with small nodules. Complete surgical resection was not possible. CONCLUSIONS: Intra-abdominal desmoplastic small round cell tumor remains an aggressive malignancy with an extremely poor prognosis. Although some response to chemotherapy may be possible, complete resection is rare, and surgical efforts are generally palliative.


Subject(s)
Carcinoma, Small Cell/pathology , Stomach Neoplasms/pathology , Adult , Carcinoma, Small Cell/drug therapy , Female , Humans , Stomach Neoplasms/drug therapy
18.
Yale J Biol Med ; 72(4): 287-93, 1999.
Article in English | MEDLINE | ID: mdl-10907779

ABSTRACT

BACKGROUND: Intra-abdominal desmoplastic small round cell tumor is a rare malignancy with a predilection for young males. Unique histological and immunocytochemical features distinguish the tumor from other members of the family of small round cell tumors of infancy and childhood. The aggressive nature of tumor spread, relative insensitivity to chemotherapy, and generally incomplete resectability result in a very poor prognosis. The authors report a case of a 39-year-old man with diffuse abdominal and pelvic involvement of intra-abdominal desmoplastic small round cell tumor treated with aggressive chemotherapy and surgery. METHODS: Computed-tomography (CT)-guided biopsy of an omental mass was performed. Histologically, discrete nests of uniform closely packed malignant cells were distributed in a background of focally desmoplastic stroma. Immunocchemistry demonstrated positivity for epithelial, mesenchymal, and neural markers. On the basis of these unique histological and immunohistochemical characteristics, the diagnosis of desmoplastic small round cell tumor was made. The patient was treated with aggressive neoadjuvant chemotherapy consisting of a high-dose alkylator -based combination regimen, followed by surgery. RESULTS: The patient had a 10 to 15 percent regression in tumor mass in response to chemotherapy. Laparotomy revealed two large omental masses, another large mass adherent to the left colon and pelvic sidewall, and diaphragmatic, peritoneal and mesenteric studding with small nodules. Complete surgical resection was not possible. CONCLUSIONS: Intra-abdominal desmoplastic small round cell tumor remains an aggressive malignancy with an extremely poor prognosis. Although some response to chemotherapy may be possible, complete resection is rare, and surgical efforts are general palliative.


Subject(s)
Abdominal Neoplasms/drug therapy , Abdominal Neoplasms/surgery , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Small Cell/drug therapy , Carcinoma, Small Cell/surgery , Abdominal Neoplasms/pathology , Adult , Biomarkers , Carcinoma, Small Cell/pathology , Chemotherapy, Adjuvant , Epithelium/metabolism , Epithelium/pathology , Humans , Laparotomy , Male , Treatment Outcome
19.
J Soc Gynecol Investig ; 1(1): 97-103, 1994.
Article in English | MEDLINE | ID: mdl-9419755

ABSTRACT

OBJECTIVE: Pharmacologic disruption of microtubule function may provide effective therapy for advanced epithelial ovarian cancer, as has been observed in clinical trials using taxol. However, the limited availability of taxol and taxol's side effects emphasize a need to develop alternative antimicrotubule agents. Estramustine (EM) inhibits binding of microtubule-associated proteins (MAPs) to microtubules, promotes microtubule disassembly, disrupts spindle formation, and induces metaphase arrest in human prostate carcinoma and glioma cells in culture. We studied the effect of EM on DNA synthesis and on the cell cycle in four human ovarian carcinoma cell lines and examined the cell lines for evidence of MAP-like immunoreactivity. METHODS: The effect of EM on DNA synthesis and on the cell cycle was determined using [3H]thymidine incorporation assays and flow cytometry, respectively. Microtubule-associated protein-like immunoreactivity was determined using monoclonal antibodies directed against MAP 1A, MAP 1B, and MAP 2(2A + 2B) for Western analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS: We demonstrated a dose-dependent inhibitory response to EM in BIXLER, DK2NMA, and SKOV3. BIX3 showed a dose-dependent inhibitory response to EM concentrations from 25 micrograms/mL to 100 micrograms/mL, but a stimulatory response at 10 micrograms/mL. Estramustine inhibited exponentially growing cells by causing mitotic arrest with subsequent accumulation of cells in G2/M phase of the cell cycle in all four cell lines. We found MAP 1A, MAP 1B, and MAP 2-like immunoreactivity in all four cells lines studied. CONCLUSIONS: These results are consistent with a MAP-microtubule mechanism of action for EM in ovarian carcinoma cells and provide reason to conduct further study of EM for potential use in the treatment of human epithelial ovarian cancer.


Subject(s)
Antineoplastic Agents, Hormonal/therapeutic use , Carcinoma/drug therapy , Cell Cycle/drug effects , DNA, Neoplasm/biosynthesis , Estramustine/therapeutic use , Ovarian Neoplasms/drug therapy , Blotting, Western , Female , Flow Cytometry , Humans , Immunohistochemistry , Microtubule-Associated Proteins/analysis , Ovarian Neoplasms/pathology , Thymidine/metabolism , Tritium , Tumor Cells, Cultured
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