Beyond single-marker analyses: mining whole genome scans for insights into treatment responses in severe sepsis.

Management of severe sepsis, an acute illness with high morbidity and mortality, suffers from the lack of effective biomarkers and largely empirical predictions of disease progression and therapeutic responses. We conducted a genome-wide association study using a large randomized clinical trial cohort to discover genetic biomarkers of response to therapy and prognosis utilizing novel approaches, including combination markers, to overcome limitations of single-marker analyses. Sepsis prognostic models were dominated by clinical variables with genetic markers less informative. In contrast, evidence for gene-gene interactions were identified for sepsis treatment responses with genetic biomarkers dominating models for predicting therapeutic responses, yielding candidates for replication in other cohorts.

VEGF levels in the alveolar compartment do not distinguish between ARDS and hydrostatic pulmonary oedema.

Although overexpression of vascular endothelial growth factor (VEGF) 165 in the lung causes pulmonary oedema, its role in human acute lung injury (ALI) is unclear. VEGF levels are reported to be lower in bronchoalveolar lavage from ALI patients compared with normals, but these studies did not include a comparably ill control group with noninflammatory pulmonary oedema. The current authors hypothesised that VEGF levels in pulmonary oedema fluid would be lower in ALI patients compared with control patients with severe hydrostatic pulmonary oedema. VEGF was measured in pulmonary oedema fluid and plasma from 56 patients with ALI and 46 controls with severe hydrostatic pulmonary oedema. Pulmonary oedema fluid levels of VEGF did not differ between patients with hydrostatic oedema (median 799 pg x mL(-1), interquartile range (IQR) 226-2,281) and ALI (median 507, IQR 0.8-1,031). Plasma levels were also the same (median 20.5 pg x mL(-1), IQR 0-152 versus 4.8, IQR 0-99.8). There was no association between plasma or oedema fluid VEGF levels and outcomes including mortality. Vascular endothelial growth factor levels in pulmonary oedema fluid were depressed both in acute lung injury and hydrostatic pulmonary oedema. The decrease in air space concentrations of vascular endothelial growth factor in acute lung injury may not be a function of the degree of lung injury, but rather may result from alveolar flooding.

Compartmentalization of vascular endothelial growth factor to the epithelial surface of the human lung.

BACKGROUND: Based on assessment of mRNA expression, the lung is a major site of expression of the vascular endothelial growth factor (VEGF) gene, largely from type II alveolar epithelial cells. With the knowledge that VEGF can function to induce vascular leak, we hypothesized that to protect the lung from pulmonary edema, the VEGF produced in the lung must be compartmentalized from the pulmonary endothelium, and thus must be compartmentalized to the surface of the respiratory epithelium. MATERIAL AND METHODS: To assess this hypothesis, we quantified the levels of VEGF in human respiratory epithelial lining fluid recovered by bronchoalveolar lavage from normal individuals. RESULTS: Strikingly, human respiratory epithelial lining fluid contains 11 +/- 5 ng/mL as quantified by ELISA, a 500-fold greater concentration than plasma (22 +/- 10 pg/mL, p < 0.0005). Western analysis of BAL fluid proteins showed the major VEGF isoform in respiratory epithelial lining fluid is VEGF165. CONCLUSIONS: With the knowledge that proteins of molecular mass like VEGF (34 to 46 kDa) slowly diffuse across the alveolar epithelium, it is likely that this high level "reservoir" of VEGF protein on the respiratory epithelial surface plays a role in normal lung endothelial biology. However, this compartmentalized VEGF reservoir may also be a "Damocles sword" poised to induce lung endothelial permeability in conditions of acute lung injury when the integrity of the alveolar epithelial barrier is breached.

Lung overexpression of the vascular endothelial growth factor gene induces pulmonary edema.

We hypothesized that the angiogenic mediator, vascular endothelial growth factor (VEGF), known to be expressed in the lung and to be capable of inducing local edema in skin, might evoke the development of lung edema if expressed in excess amounts. To test this hypothesis, we developed an in vivo model of VEGF overexpression in the lung on the basis of delivery to the respiratory epithelium of the VEGF165 complementary DNA by an E1(-) adenovirus vector (AdVEGF165). Administration of AdVEGF165 by the intratracheal route (10(9) plaque-forming units [pfu]) to C57Bl/6 mice showed increased expression of VEGF messenger RNA in lung tissue by Northern analysis. Overexpression of VEGF protein in the lung at Days 1 to 10 was confirmed by enzyme-linked immunosorbent assay. Intratracheal administration of AdVEGF165 resulted in a dose-dependent increase in lung wet/dry weight ratios over time, lung histology showed widespread intra- alveolar edema, and pulmonary capillary permeability was significantly increased as quantified by the Evans blue dye assay and [(131)I]albumin permeability. To confirm the specificity of these observations, mice were pretreated with intranasal administration of an adenovirus vector expressing a truncated soluble form of the VEGF receptor flt-1 (Adsflt). Adsflt (10(9) pfu) pretreatment completely abrogated the increased lung wet/dry weight ratio caused by AdVEGF165 administration, whereas an identical adenovirus vector with an irrelevant transgene had no effect upon subsequent AdVEGF165-induced pulmonary edema. Together, these data suggest that overexpression of VEGF in the lung may be one mechanism of increased pulmonary vascular permeability in the early stages of acute lung injury.

Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus.

We sought to evaluate the ability of an E1(-), E3(-) adenovirus (Ad) vector (Ad(GV)CFTR.10) to transfer the normal human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the airway epithelium of individuals with cystic fibrosis (CF). We administered Ad(GV)CFTR.10 at doses of 3 x 10(6) to 2 x 10(9) plaque-forming units over 9 months by endobronchial spray to 7 pairs of individuals with CF. Each 3-month cycle, we measured vector-derived versus endogenous CFTR mRNA in airway epithelial cells prior to therapy, as well as 3 and 30 days after therapy. The data demonstrate that (a) this strategy appears to be safe; (b) after the first administration, vector-derived CFTR cDNA expression in the CF airway epithelium is dose-dependent, with greater than 5% endogenous CFTR mRNA levels at the higher vector doses; (c) expression is transient, lasting less than 30 days; (d) expression can be achieved with a second administration, but only at intermediate doses, and no expression is observed with the third administration; and (e) the progressive lack of expression with repetitive administration does not closely correlate with induction of systemic anti-Ad neutralizing antibodies. The major advantage of an Ad vector is that it can deliver sufficient levels of CFTR cDNA to the airway epithelium so that CFTR expression protects the lungs from the respiratory manifestations of CF. However, this impressive level of expression is linked to the challenging fact that expression is limited in time. Although this can be initially overcome by repetitive administration, unknown mechanisms eventually limit this strategy, and further repetitive administration does not lead to repetitive expression.

Expression and use of human immunodeficiency virus type 1 coreceptors by human alveolar macrophages.

Human immunodeficiency virus type 1 (HIV-1) requires, in addition to CD4, coreceptors of the CC or CXC chemokine families for productive infection of T cells and cells of the monocyte-macrophage lineage. Based on the hypothesis that coreceptor expression on alveolar macrophages (AM) may influence HIV-1 infection of AM in the lung, this study analyzes the expression and utilization of HIV-1 coreceptors on AM of healthy individuals. AM were productively infected with five different primary isolates of HIV-1. Levels of surface expression of CCR5, CXCR4, and CD4 were low compared to those of blood monocytes, but CCR3 was not detectable. mRNA for CCR5, CXCR4, CCR2, and CCR3 were all detectable, but to varying degrees and with variability among donors. Expression of CCR5, CXCR4, and CCR2 mRNA was downregulated following stimulation with lipopolysaccharide (LPS). In contrast, secretion of the chemokines RANTES, MIP-1alpha, and MIP-1beta was upregulated with LPS stimulation. Interestingly, HIV-1 replication was diminished following LPS stimulation. Infection of AM with HIV-1 in the presence of the CC chemokines demonstrated blocking of infection. Together, these studies demonstrate that AM can be infected by a variety of primary HIV-1 isolates, AM express a variety of chemokine receptors, the dominant coreceptor used for HIV entry into AM is CCR5, the expression of these receptors is dependent on the state of activation of AM, and the ability of HIV-1 to infect AM may be modulated by expression of the chemokine receptors and by chemokines per se.

Modification of the genetic program of human alveolar macrophages by adenovirus vectors in vitro is feasible but inefficient, limited in part by the low level of expression of the coxsackie/adenovirus receptor.

Robust expression of genes transferred by adenovirus (Ad) vectors depends upon efficient entry of vectors into target cells. Cells deficient in the coxsackie/adenovirus receptor (CAR) are difficult targets for Ad-mediated gene transfer. We hypothesized that low levels of CAR expression may be responsible, in part, for the relative inefficiency of Ad-mediated gene transfer to human alveolar macrophages (AMs). CAR gene expression was detected in human AMs by reverse transcription-polymerase chain reaction and at low levels by Northern analysis. Indirect immunofluorescence showed specific, low-intensity surface staining for CAR, but at levels below those found on the positive-control A549 human lung epithelial cell line. Consistent with this, AMs expressed Ad vector transgenes 100 to 1,000-fold less efficiently than A549 cells, as assessed using the beta-galactosidase reporter (chemiluminescence assay) and green fluorescent protein (fluorescence microscopy and flow cytometry). At high multiplicity of infection, AMs from an HIV+ individual could be transduced with an AdIFNgamma vector to secrete detectable human interferon-gamma. Ad transgene expression by AMs was blocked by capsid fiber protein, suggesting that CAR is required in the pathway for productive Ad entry into alveolar macrophages. To confirm that Ad transgene expression by AMs is limited by low levels of CAR expression, cells were infected with an Ad vector containing the CAR complementary DNA (cDNA). Enhanced expression of CAR protein was demonstrated by indirect immunofluorescence, and the CAR cDNA-transduced cells showed 5-fold enhancement of subsequent Ad transgene expression. These observations demonstrate that human AMs can be targets for Ad-mediated gene transfer, but that efficiency of transgene expression is limited, at least in part, by low levels of CAR expression.

Selective expansion of alveolar macrophages in vivo by adenovirus-mediated transfer of the murine granulocyte-macrophage colony-stimulating factor cDNA.

Based on the hypothesis that genetic modification of freshly isolated alveolar macrophages (AM) with the granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA would induce AM to proliferate, this study focuses on the ability of adenoviral (Ad) vectors to transfer and efficiently express the murine (m) GM-CSF cDNA in murine AM with consequent expansion in the number of AM in vitro and in vivo. To demonstrate that an Ad vector can effectively transfer and express genes in AM, murine AM recovered by bronchoalveolar lavage from the lung of Balb/c mice were infected with an Ad vector coding for green fluorescent protein (GFP) in vitro and expressed GFP in a dose-dependent fashion. Infection of AM with an Ad vector containing an expression cassette coding for mGM-CSF led to GM-CSF expression and to AM proliferation in vitro. When AM infected with AdGFP were returned to the respiratory tract of syngeneic recipient mice, GFP-expressing cells could still be recovered by bronchoalveolar lavage 2 weeks later. In vitro infection of AM with AdmGM-CSF and subsequent transplantation of the genetically modified AM to the lungs of syngeneic recipients led to GM-CSF expression in vivo. Strikingly, the AM recovered by lavage 5 weeks after transplantation demonstrated an increased rate of proliferation, and the total number of alveolar macrophages was 1. 9-fold greater than controls. Importantly, the increase in the numbers of AM was selective (ie, other inflammatory cell numbers were unchanged), and there was no modification to the lung architecture. Thus, it is feasible to genetically modify AM with Ad vectors and to use this strategy to modify the behavior of AM in vivo. Based on the importance of AM in the primary defense of the respiratory epithelial surface, this strategy may be useful in enhancing pulmonary defenses in immunodeficiency states.

Pulmonary complications in cancer patients.

Pulmonary complications of cancer and cancer therapy represent a broad spectrum of disease. Early diagnosis and treatment are essential to achieve an optimal outcome.

Altered cholesterol trafficking in herpesvirus-infected arterial cells. Evidence for viral protein kinase-mediated cholesterol accumulation.

Herpesvirus infection of arterial smooth muscle cells has been shown to cause cholesteryl ester (CE) accumulation. However, the effects of human herpes simplex virus type 1 (HSV-1) infection on cholesterol binding and internalization, intracellular metabolism, and efflux have not been evaluated. In addition, the effects of viral infection on signal transduction pathways that impact upon cholesterol metabolism have not been studied. We show in studies reported herein that HSV-1 infection of arterial smooth muscle cells enhances low density lipoprotein (LDL) binding and uptake which parallels an increase in LDL receptor steady state mRNA levels and transcription of the LDL receptor gene. HSV-2 also increases CE synthesis and 3-hydroxy- 3-methylglutaryl-CoA reductase activity but concomitantly reduces CE hydrolysis and cholesterol efflux. Interestingly, this viral infection was associated with a time-dependent decrease in protein kinase A activity and an increase in viral-induced protein kinase (VPK) activity commensurate with the accumulation of esterified cholesterol. The relationship between increased VPK activity and alterations in CE accumulation in virally infected cells was explored using an HSV-1 VPK- mutant in which the portion of the HSV-1 genome encoding VPK had been deleted. Cholesteryl ester accumulation was significantly increased (> 50-fold) in HSV-1-infected cells compared to uninfected cells. However, the HSV-1 VPK- mutant had no significant effect on CE accumulation. The relationship between VPK activity and these alterations in cholesterol metabolism was further supported by the observation that staurosporine and calphostin C (protein kinase inhibitors) reduced protein kinase activity in HSV-1-infected cells. These results suggest several potential mechanisms by which alterations in kinase activities in response to HSV-1 infection of vascular cells may alter cholesterol trafficking processes that eventually lead to CE accumulation.

Aspiration emergencies.

Aspiration of foreign material into the lungs can represent a medical emergency requiring timely interventions to assure a favorable outcome. Establishment of a patent airway and maintenance of adequate oxygenation are the initial requirements for successful treatment of all types of aspiration emergencies. The nature of the aspirated material dictates further interventions and potential outcome.

Developmentally regulated herpesvirus plaque formation in arterial smooth muscle cells.

Previous studies of age-related susceptibility to viral infection have focused largely on the effects of aging on the immune response. Little attention has been given to age-related changes in the infectivity of target cells. We show here a fourfold greater plating efficiency of herpes simplex virus type 1 (HSV-1) for cultured vascular smooth muscle cells derived from adult rats compared with cells from genetically identical pup rats. The difference in plating efficiency appeared to be due to differences in initial attachment of the virion to the cell surface. There were no differences in the rate of viral entry or the efficiency of viral replication at high multiplicities of infection and no resistant "subpopulation" of pup cells. The pup cells did not release a soluble inhibitor of infection. Infection in both cell types was inhibited similarly by basic fibroblast growth factor (bFGF). Although adult cells exhibited a more vigorous mitogenic response to bFGF than did pup cells, binding studies did not demonstrate significant differences in the binding of bFGF to the cell surface, suggesting that differential expression of high-affinity FGF receptors could not be correlated with the difference in infectivity. We speculate that differences in the distribution of heparan sulfate in the cell surface, which serves as the initial attachment site for HSV-1, may explain the observed differences in plating efficiency. Since age is a risk factor for the development of atherosclerosis, these results have potential implications for susceptibility of the vasculature to herpesviral infections as a function of the development of the vessel wall.

Mediation of virion penetration into vascular cells by association of basic fibroblast growth factor with herpes simplex virus type 1.

Herpes simplex virus type-1 (HSV-1) is a ubiquitous pathogen that is associated with considerable morbidity in the general population. Although it is known that the virion uses a basic fibroblast growth factor (FGF) receptor to penetrate vascular cells, it is not known how the viral particle recognizes and binds to this cell surface protein. Here we report that an immunoreactive basic FGF-like protein is associated with the viral particle and that this association appears responsible for viral uptake. Accordingly, HSV-1 infection of Swiss 3T3 cells stimulates the tyrosine phosphorylation of the specific substrate that characterizes the initial cellular response to basic FGF. Antibodies to basic FGF prevent this phosphorylation and inhibit HSV-1 uptake. Because no basic FGF sequence is found in the HSV-1 genome, a model for the infection for some target cells is presented whereby the viral particle uses host cell-derived basic FGF to ensure subsequent infectivity of newly replicated virus.

Fibroblast growth factor receptor is a portal of cellular entry for herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) is a ubiquitous pathogen responsible for considerable morbidity in the general population. The results presented herein establish the basic fibroblast growth factor (FGF) receptor as a means of entry of HSV-1 into vertebrate cells. Inhibitors of basic FGF binding to its receptor and competitive polypeptide antagonists of basic FGF prevented HSV-1 uptake. Chinese hamster ovary (CHO) cells that do not express FGF receptors are resistant to HSV-1 entry; however, HSV-1 uptake is dramatically increased in CHO cells transfected with a complementary DNA encoding a basic FGF receptor. The distribution of this integral membrane protein in vivo may explain the tissue and cell tropism of HSV-1.

Inhibition of proteoglycan synthesis in human endothelial cells after infection with herpes simplex virus type 1 in vitro.

The effects of herpes simplex virus type 1 (HSV-1) infection on proteoglycan synthesis by human endothelial cells were studied as a model of endothelial cell injury. Confluent cultures of early passage endothelial cells from human umbilical vein were infected with HSV-1 at multiplicities of infection from 0.001 to 1.0. HSV-1 infection produced a dose-dependent inhibition of total proteoglycan synthesis of up to 85%. Although there was a 2- to 3-fold increase in the quantity of virus necessary to cause 50% inhibition of heparan sulfate compared to chondroitin/dermatan sulfate proteoglycan, the inhibition was relatively parallel, even up to high virus doses. There was no inhibition of an undersulfated heparan sulfate proteoglycan that contained glycosaminoglycan chains shorter than the predominant species. The results indicate that HSV-1 infection of human endothelial cells produces complex effects on host-cell metabolism. The viral-induced changes in proteoglycan metabolism may influence cell-matrix interactions and lead to altered vessel wall function.

In vitro delayed hypersensitivity granuloma formation: development of an antigen-coated bead model.

Previously, we have described an in vitro model of granulomatous hypersensitivity around Schistosoma mansoni eggs in both the murine model of schistosomiasis and in human schistosomiasis. These studies describe a new model of in vitro granuloma formation that complexes soluble egg antigen from S. mansoni eggs, a partially purified protein derivative of Mycobacterium tuberculosis (PPD), or bovine serum albumin to carrier beads. Ultrastructural and morphologic evaluations demonstrate that there are initial macrophage interactions, followed by the recruitment of antigen-specific T cells that interact with and recruit macrophages, lymphocytes, granulocytes, and fibroblasts. Finally, there is a stage of granulomatous organization involving fibroblast proliferation and collagen deposition. The in vitro reactivity, defined by a quantitative granuloma index, correlates with in vivo granulomas around S. mansoni eggs in the livers of infected cell donor animals. In vitro granuloma formation against PPD-coated beads correlated with delayed cutaneous hypersensitivity against PPD, which was judged by footpad swelling. The reactions demonstrate antigenic specificity and were intrinsically modulated in a manner that is analogous to that previously shown with the in vitro egg granuloma model. This model of in vitro granuloma formation promises to be a useful tool for elucidating mechanisms of cellular immunity and regulation.