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W Conrad Liles

Publications and source records attributed to W Conrad Liles.

At least 19 recordsLinked to original sources
PubMed2006

Simultaneous host and parasite expression profiling identifies tissue-specific transcriptional programs associated with susceptibility or resistance to experimental cerebral malaria.

BACKGROUND: The development and outcome of cerebral malaria (CM) reflects a complex interplay between parasite-expressed virulence factors and host response to infection. The murine CM model, Plasmodium berghei ANKA (PbA), which simulates many of the features of human CM, provides an excellent system to study this host/parasite interface. We designed "combination" microarrays that concurrently detect genome-wide transcripts of both PbA and mouse, and examined parasite and host transcriptional programs during infection of CM-susceptible (C57BL/6) and CM-resistant (BALB/c) mice. RESULTS: Analysis of expression data from brain, lung, liver, and spleen of PbA infected mice showed that both host and parasite gene expression can be examined using a single microarray, and parasite transcripts can be detected within whole organs at a time when peripheral blood parasitemia is low. Parasites display a unique transcriptional signature in each tissue, and lung appears to be a large reservoir for metabolically active parasites. In comparisons of susceptible versus resistant animals, both host and parasite display distinct, organ-specific transcriptional profiles. Differentially expressed mouse genes were related to humoral immune response, complement activation, or cell-cell interactions. PbA displayed differential expression of genes related to biosynthetic activities. CONCLUSION: These data show that host and parasite gene expression profiles can be simultaneously analysed using a single "combination" microarray, and that both the mouse and malaria parasite display distinct tissue- and strain-specific responses during infection. This technology facilitates the dissection of host-pathogen interactions in experimental cerebral malaria and could be extended to other disease models.

Animals↗
PubMed2006

cDNA microarray analysis reveals fundamental differences in the expression profiles of primary human monocytes, monocyte-derived macrophages, and alveolar macrophages.

We report the systematic use of large-scale cDNA microarrays to study the gene expression profiles of primary human peripheral blood monocytes (MONO) in comparison with in vitro-differentiated, M-CSF-induced MONO-derived macrophages (MAC) and primary human alveolar MAC (AM), obtained by bronchoalveolar lavage from the lungs of normal volunteers. These studies revealed large-scale differences in the gene expression profile between both MAC types (MAC and AM) and MONO. In addition, large differences were observed in the gene expression profiles of the two MAC types. Specifically, 21% of genes on the array (2904 out of 13,582) were differentially expressed between AM and MONO, and 2229 out of 13,583 probes were differentially expressed between MAC and AM. Our expression data show remarkable differences in gene expression between different MAC subpopulations and emphasize the heterogeneity of different MAC populations. This study underscores the need to scrutinize models of MAC biology for relevance to specific disease processes.

Adult↗
PubMed2006

Mechanical ventilation induces inflammation, lung injury, and extra-pulmonary organ dysfunction in experimental pneumonia.

Mechanical ventilation (MV) is frequently employed for the management of critically ill patients with respiratory failure. A major complication of mechanical ventilation (MV) is the development of ventilator-associated pneumonia (VAP), in which Staphylococcus aureus is a prominent pathogen. Moreover, previous studies suggest that MV may be an important cofactor in the development of acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS). S. aureus pulmonary infection was induced in spontaneously breathing mice (C57Bl/6) or mechanically ventilated mice to determine whether MV contributes to the development of ALI and/or systemic inflammation. The combination of MV and bacteria significantly increased the influx of neutrophils into bronchoalveolar lavage fluid (BALF), augmented pulmonary production of the proinflammatory cytokines KC, MIP-2, TNF-alpha, and IL-6, and increased alveolar-capillary permeability to proteins. MV also induced proinflammatory cytokine expression in peripheral blood, associated with extrapulmonary hepatic and renal dysfunction. Surprisingly, bacterial clearance in the lungs and extrapulmonary bacterial dissemination was not affected by MV. These data indicate that MV exacerbates both pulmonary and systemic inflammation in response to bacteria and contributes to the pathogenesis of both ALI and the multiple organ dysfunction syndrome, without necessarily affecting bacterial clearance or extra-pulmonary bacterial dissemination.

Animals↗
PubMed2006

Mechanical ventilation interacts with endotoxemia to induce extrapulmonary organ dysfunction.

INTRODUCTION: Multiple organ dysfunction syndrome (MODS) is a common complication of sepsis in mechanically ventilated patients with acute respiratory distress syndrome, but the links between mechanical ventilation and MODS are unclear. Our goal was to determine whether a minimally injurious mechanical ventilation strategy synergizes with low-dose endotoxemia to induce the activation of pro-inflammatory pathways in the lungs and in the systemic circulation, resulting in distal organ dysfunction and/or injury. METHODS: We administered intraperitoneal Escherichia coli lipopolysaccharide (LPS; 1 microg/g) to C57BL/6 mice, and 14 hours later subjected the mice to 6 hours of mechanical ventilation with tidal volumes of 10 ml/kg (LPS + MV). Comparison groups received ventilation but no LPS (MV), LPS but no ventilation (LPS), or neither LPS nor ventilation (phosphate-buffered saline; PBS). RESULTS: Myeloperoxidase activity and the concentrations of the chemokines macrophage inflammatory protein-2 (MIP-2) and KC were significantly increased in the lungs of mice in the LPS + MV group, in comparison with mice in the PBS group. Interestingly, permeability changes across the alveolar epithelium and histological changes suggestive of lung injury were minimal in mice in the LPS + MV group. However, despite the minimal lung injury, the combination of mechanical ventilation and LPS resulted in chemical and histological evidence of liver and kidney injury, and this was associated with increases in the plasma concentrations of KC, MIP-2, IL-6, and TNF-alpha. CONCLUSION: Non-injurious mechanical ventilation strategies interact with endotoxemia in mice to enhance pro-inflammatory mechanisms in the lungs and promote extra-pulmonary end-organ injury, even in the absence of demonstrable acute lung injury.

Animals↗
PubMed2005

Computational identification of key biological modules and transcription factors in acute lung injury.

RATIONALE: Mechanical ventilation augments the acute lung injury (ALI) caused by bacterial products. The molecular pathogenesis of this synergistic interaction remains incompletely understood. OBJECTIVE: We sought to develop a computational framework to systematically identify gene regulatory networks activated in ALI. METHODS: We have developed a mouse model in which the combination of mechanical ventilation and intratracheal LPS produces significantly more injury to the lung than either insult alone. We used global gene ontology analysis to determine overrepresented biological modules and computational transcription factor analysis to identify putative regulatory factors involved in this model of ALI. RESULTS: By integrating expression profiling with gene ontology and promoter analysis, we constructed a large-scale regulatory modular map of the important processes activated in ALI. This map assigned differentially expressed genes to highly overrepresented biological modules, including "defense response," "immune response," and "oxidoreductase activity." These modules were then systematically incorporated into a gene regulatory network that consisted of putative transcription factors, such as IFN-stimulated response element, IRF7, and Sp1, that may regulate critical processes involved in the pathogenesis of ALI. CONCLUSIONS: We present a novel, unbiased, and powerful computational approach to investigate the synergistic effects of mechanical ventilation and LPS in promoting ALI. Our methodology is applicable to any expression profiling experiment involving eukaryotic organisms.

Animals↗
PubMed2005

Fas-mediated acute lung injury requires fas expression on nonmyeloid cells of the lung.

Fas (CD95) is a membrane surface receptor, which, in the lungs, is expressed in macrophages, neutrophils, and epithelial cells. In mice, Fas activation leads to a form of lung injury characterized by increased alveolar permeability. We investigated whether Fas-mediated lung injury occurs primarily as a result of Fas activation in myeloid cells (such as macrophages) or in nonmyeloid cells (such as epithelial cells). Chimeric mice lacking Fas in either myeloid or nonmyeloid cells were generated by transplanting marrow cells from lpr mice (which lack Fas) into lethally irradiated C57BL/6 mice (MyFas(-) group) or vice versa (MyFas(+) group). Additional mice transplanted with marrow cells from their same strain served as controls (Fas(+) ctr and Fas(-) ctr groups). Sixty days after transplantation, the mice received intratracheal instillations of the Fas-activating mAb Jo2 (n = 10/group), or an isotype control Ab (n = 10/group), and were euthanized 24-h later. Only animals expressing Fas in nonmyeloid cells (Fas(+) ctr and MyFas(-)) showed significant increases in lung neutrophil content and in alveolar permeability. These same mice showed tissue evidence of lung injury and caspase-3 activation in cells of the alveolar walls. Despite differences in the neutrophilic response and lung injury, there was no statistical difference in the lung cytokine concentrations (KC and MIP-2) among groups. We conclude that Fas-mediated lung injury requires expression of Fas on nonmyeloid cells of the lungs. These findings suggest that the alveolar epithelium is the primary target of Fas-mediated acute lung injury, and demonstrate that apoptotic processes may be associated with neutrophilic inflammation.

Animals↗
PubMed2005

Modulation of lipopolysaccharide-induced gene transcription and promotion of lung injury by mechanical ventilation.

Mechanical ventilation (MV) with tidal volumes of 10-12 ml/kg is considered safe in the absence of acute lung injury (ALI). However, recent studies show that, when lung injury is already present, tidal volumes of this magnitude increase inflammation and injury in the lungs. We hypothesized that MV with tidal volumes of 10-ml/kg can also function as a cofactor in the initiation of ALI by modulating the transcriptional response to bacterial products. To test this hypothesis, we developed a mouse model in which MV did not independently cause inflammation or injury but augmented the inflammatory response to low-dose aspirated LPS and promoted development of ALI. We analyzed gene expression in lungs from 24 mice assigned to four different groups: control, MV only, intratracheal LPS only, and MV + LPS. There were twice as many differentially regulated genes in the MV + LPS group compared with the LPS-only group and 10 times as many differentially regulated genes compared with the MV-only group. For genes up-regulated by LPS treatment alone, the addition of MV further augmented expression. Cytokine concentrations in bronchoalveolar lavage fluid and tissue distribution of an intracellular protein, GADD45-gamma, correlated with mRNA levels. We conclude that MV with conventional tidal volumes enhanced the transcriptional response to LPS and promoted development of ALI.

Animals↗
PubMed2005

NADPH oxidase restrains the matrix metalloproteinase activity of macrophages.

Matrix metalloproteinases (MMPs) regulate numerous functions in normal and disease processes; thus, irreversibly blocking their activity is a key step in regulating MMP catalysis. We previously showed in vitro that oxidizing intermediates generated by phagocytes inactivate MMPs by modifying specific amino acids. To assess whether this mechanism operates in vivo, we focused on MMP-12, a macrophage-specific MMP known to mediate emphysema in mouse models. We found that mice lacking gp91(phox), a phagocyte-specific component of the NADPH oxidase, developed extensive, spontaneous emphysematous destruction of their peripheral air spaces, whereas mice deficient in both NADPH oxidase and MMP-12 were protected from spontaneous emphysema. Although gp91(phox)-null and wild-type macrophages produced equivalent levels of MMP-12 protein, the oxidant-deficient cells had greater MMP-12 activity than wild-type macrophages. These findings indicate that reactive intermediates provide a physiological mechanism to protect tissues from excessive macrophage-mediated damage during inflammation.

Animals↗
PubMed2005

Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist.

Improving approaches for hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) mobilization is clinically important because increased numbers of these cells are needed for enhanced transplantation. Chemokine stromal cell derived factor-1 (also known as CXCL12) is believed to be involved in retention of HSCs and HPCs in bone marrow. AMD3100, a selective antagonist of CXCL12 that binds to its receptor, CXCR4, was evaluated in murine and human systems for mobilizing capacity, alone and in combination with granulocyte colony-stimulating factor (G-CSF). AMD3100 induced rapid mobilization of mouse and human HPCs and synergistically augmented G-CSF-induced mobilization of HPCs. AMD3100 also mobilized murine long-term repopulating (LTR) cells that engrafted primary and secondary lethally-irradiated mice, and human CD34(+) cells that can repopulate nonobese diabetic-severe combined immunodeficiency (SCID) mice. AMD3100 synergized with G-CSF to mobilize murine LTR cells and human SCID repopulating cells (SRCs). Human CD34(+) cells isolated after treatment with G-CSF plus AMD3100 expressed a phenotype that was characteristic of highly engrafting mouse HSCs. Synergy of AMD3100 and G-CSF in mobilization was due to enhanced numbers and perhaps other characteristics of the mobilized cells. These results support the hypothesis that the CXCL12-CXCR4 axis is involved in marrow retention of HSCs and HPCs, and demonstrate the clinical potential of AMD3100 for HSC mobilization.

Animals↗
PubMed2005

Role of caspases in death and survival of the plaque macrophage.

This review considers the role of macrophage cell death in formation of the necrotic core and in plaque progression, and lists many of the possible mediators of macrophage cell death. Among these, perhaps the most cited toxic agent is oxidized low-density lipoprotein (oxLDL). Whereas oxLDL can kill macrophage, and whereas the form of death is morphologically apoptotic, caspase inhibitors appear to be ineffective in preventing death. This finding is consistent with recent literature showing how the canonical caspase pathways are used for physiological cellular functions other than cell death. Plaque macrophages appear to be among the cells with this nonapoptotic signaling function for activated caspases. In many of the other cell types, caspase activation appears to play a critical role in cell differentiation. We discuss possible functions of plaque macrophage using the nondeath caspase pathway. Recent literature shows that physiological and developmental functions of many cell types require active caspases without progressing to cell death. We discuss the role of macrophage cell death in plaque progression, possible mediators of macrophage cell death, and the possible functions of plaque macrophage using the nondeath caspase pathway.

Animals↗
PubMed2005

Blockade of the Fas/FasL system improves pneumococcal clearance from the lungs without preventing dissemination of bacteria to the spleen.

BACKGROUND: The Fas/FasL system is both proapoptotic and proinflammatory. FasL is inhibited by decoy receptor-3 (DcR3), a naturally occurring decoy receptor. We determined the effects of systemic blockade of the Fas/FasL system by a DcR3 analog (DcR3-a) in mice with pneumococcal pneumonia. METHODS: Streptococcus pneumoniae (7.2 x 105 or 1.9 x 107 cfu/mL) was instilled intratracheally into untreated C57Bl/6 mice, C57Bl/6 mice treated with DcR3-a, or Fas-deficient lpr mice, and the mice were studied 48 h later. RESULTS: After instillation of the lower bacterial dose, disruption of the Fas/FasL system by either DcR3-a or the lpr mutation resulted in improved clearance of bacteria in the lungs (mean +/- SE, 4.6+/-2.1 x 10(6) and 3.5 +/- 1.6 x 10(6) cfu/lung, respectively, vs. 21.9+/-9.3 x 10(6) cfu/lung in untreated C57Bl/6 mice; P<.05) and decreased percentage of polymorphonuclear neutrophils in bronchoalveolar lavage fluid (mean +/- SE, 19.3%+/-9.5% and 20.2%+/-7.8%, respectively, vs. 55.0%+/-12.2% in untreated C57Bl/6 mice; P<.05). These changes were associated with decreased lung concentrations of the proinflammatory cytokines tumor necrosis factor- alpha and macrophage inflammatory protein-2 and with a decrease in apoptotic cells in the alveolar walls. CONCLUSION: Blockade of the Fas/FasL system by DcR3-a in the lungs improves clearance of bacteria in mice with pneumococcal pneumonia.

Amino Acid Substitution↗
PubMed2005

Hyper-IgE syndrome is not associated with defects in several candidate toll-like receptor pathway genes.

The genetic basis of hyper-IgE syndrome (HIES), also known as Job syndrome, a primary immunodeficiency associated with recurrent skin and pulmonary infections, is unknown. We hypothesized that HIES is due to a defect in the Toll-like receptor signaling pathway. We used a whole blood cytokine assay to compare inflammatory responses to stimulation with specific Toll-like receptor (TLR) pathway agonists in four individuals with HIES and nine healthy controls. Production of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and IL-12 was not impaired in response to stimulation with lipopolysaccharide, peptidoglycan, zymosan, lipoteichoic acid, Staphylococcus aureus, Escherichia coli, or Streptococcus pneumoniae. Interferon (IFN)-gamma was reduced in HIES subjects in response to each of these stimuli. We sequenced several candidate genes from the TLR pathway in HIES individuals to determine whether any mutations were associated with this syndrome. No novel mutations or polymorphisms were found in the coding regions of TLR1, TLR2, TLR6, MyD88, or TRAF6. In summary, although HIES individuals had an IFN-gamma secretion defect, they also produced normal levels of several TLR-regulated proinflammatory cytokines. No unique mutations or polymorphisms were observed in several candidate genes from the TLR pathway. Our studies do not support a role for a defective TLR response in HIES individuals.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Effective storage of granulocytes collected by centrifugation leukapheresis from donors stimulated with granulocyte-colony-stimulating factor.

BACKGROUND: Donor stimulation with granulocyte-colony-stimulating factor (G-CSF) has increased the number of neutrophils (PMNs) that can be collected for granulocyte transfusion therapy. Clinical utility, however, has been limited by the inability to store functional PMNs ex vivo. This study was conducted to determine whether granulocyte products from G-CSF-stimulated donors could be effectively stored at reduced temperature (22 degrees C vs. 10 degrees C) with maintenance of functional properties in vitro and in vivo. STUDY DESIGN AND METHODS: Nine normal subjects received G-CSF (600 microg subcutaneously) 12 hours before centrifugation leukapheresis. Granulocyte products were divided and stored for 24 and 48 hours under four conditions: 1) 22 degrees C; 2) 22 degrees C, with supplemental G-CSF (100 ng/mL); 3) 10 degrees C; and 4) 10 degrees C, with supplemental G-CSF. Functional PMN activity during ex vivo storage was assessed in vitro and in vivo by the skin-window technique for granulocytes stored at 10 degrees C for 24 hours. RESULTS: Surface expression of CD11b/CD18, CD14, CD16, CD32, and CD64 was maintained during 48-hour storage at reduced temperature. Inducible respiratory burst activity, bactericidal activity, and fungicidal activity were preserved during storage for 48-hour storage at 10 degrees C. Proinflammatory cytokine production was decreased in product stored at 10 degrees C. Supplemental G-CSF ex vivo did not substantially improve functional activity during storage. After storage at 10 degrees C for 24 hours, in vitro chemotactic potential was maintained, and transfused granulocytes retained capacity to circulate and migrate appropriately in vivo. CONCLUSIONS: Granulocyte product collected by centrifugation leukapheresis from G-CSF-stimulated donors can be effectively stored at subphysiologic temperature for 24 hours with preservation of functional activity. Storage at 10 degrees C appears to be slightly superior to storage at 22 degrees C.

Adult↗
PubMed2005

Augmented mobilization and collection of CD34+ hematopoietic cells from normal human volunteers stimulated with granulocyte-colony-stimulating factor by single-dose administration of AMD3100, a CXCR4 antagonist.

BACKGROUND: AMD3100, a selective antagonist of CXCR4, rapidly mobilizes CD34+ hematopoietic progenitor cells (HPCs) from marrow to peripheral blood with minimal side effects. STUDY DESIGN AND METHODS: To further investigate potential clinical utility of AMD3100 for CD34+ cell mobilization and collection, a Phase I study in normal volunteers was performed examining single-dose administration of AMD3100 alone and in combination with a standard 5-day granulocyte-colony-stimulating factor (G-CSF) regimen. RESULTS: AMD3100 (160 microg/kg x 1 on Day 5) significantly increased both G-CSF-stimulated (10 microg/kg/day) mobilization of CD34+ cells (3.8-fold) and leukapheresis yield of CD34+ cells. Moreover, collection of CD34+ cells was comparable between individuals mobilized by a single-dose regimen of AMD3100 (240 microg/kg) and individuals mobilized with a 5-day regimen of G-CSF. AMD3100-mobilized leukapheresis products contained significantly greater numbers of T and B cells compared to G-CSF-stimulated leukapheresis products. CONCLUSION: These findings indicate that AMD3100 can be used alone or as an adjunct to G-CSF to mobilize cells for HPC transplantation.

Anti-HIV Agents↗
PubMed2005

Chlamydia pneumoniae augments the oxidized low-density lipoprotein-induced death of mouse macrophages by a caspase-independent pathway.

Chlamydia pneumoniae is a common respiratory pathogen that is associated with an increased risk of cardiovascular disease. However, the mechanisms by which C. pneumoniae contributes to cardiovascular disease have not been determined yet. C. pneumoniae infection may accelerate the death of cells within atherosclerotic lesions and contribute to the formation of unstable lesions. To test this hypothesis, the impact of C. pneumoniae infection on the death of lipid-loaded mouse macrophages was investigated. It was observed that RAW 264.7 cells are highly susceptible to the toxic effects of oxidized low-density lipoprotein (LDL) and exhibit markers of cell death within 24 h of treatment with as little as 5 microg/ml oxidized LDL. Subsequent infection with either live C. pneumoniae or heat-killed or UV-inactivated C. pneumoniae at a low multiplicity of infection for 24 to 72 h stimulated both additional binding of annexin V and the uptake of propidium iodide. Thus, C. pneumoniae augments the effects of oxidized LDL on cell death independent of a sustained infection. However, unlike oxidized LDL, C. pneumoniae infection does not activate caspase 3 or induce formation of the mitochondrial transition pore or the fragmentation of DNA, all of which are classical markers of apoptosis. Furthermore, primary bone marrow macrophages isolated from mice deficient in Toll-like receptor 2 (TLR-2) but not TLR-4 are resistant to C. pneumoniae-induced death. These data suggest that C. pneumoniae kills cells by a caspase-independent pathway and that the process is potentially mediated by activation of TLR-2.

Animals↗
PubMed2005

Effect of Chlamydia pneumoniae on cellular ATP content in mouse macrophages: role of Toll-like receptor 2.

Chlamydiae are obligate intracellular gram-negative bacteria and are dependent on the host cell for ATP. Thus, chlamydial infection may alter the intracellular levels of ATP and affect all energy-dependent processes within the cell. We have shown that both live C. pneumoniae and inactivated C. pneumoniae induce markers of cell death prior to completion of the bacterial growth cycle. As depletion of ATP could account for the observed increase in cell death, the effects of C. pneumoniae on ATP concentrations within mouse macrophages were investigated. Live, heat-killed, and UV-inactivated C. pneumoniae cultures (at multiplicities of infection [MOIs] of 0.01, 0.1, and 1.0) were incubated with mouse bone marrow macrophages isolated from C57BL/6J mice and mice deficient in Toll-like receptors. Treatment of the macrophages with both live and inactivated C. pneumoniae increased the ATP content of the cells. In cells infected with live C. pneumoniae, the increase was inversely proportional to the MOI. In cells treated with inactivated C. pneumoniae, the increase in ATP content was smaller than that induced by infection with live organisms and was proportional to the MOI. The increase in ATP content early in the developmental cycle was independent of the growth of C. pneumoniae, while sustained induction required live organisms. The capacity of C. pneumoniae to increase the ATP content was ablated in macrophages deficient in expression of either Toll-like receptor 2 or the Toll-like receptor accessory protein MyD88. In contrast, no effect was observed in macrophages lacking expression of Toll-like receptor 4.

Adenosine Triphosphate↗