James P. Maloney

North American Summit on Aspiration in the Critically Ill Patient: Consensus Statement

Aspiration is the leading cause of pneumonia in the intensive care unit and the most serious complication of enteral tube feeding (ETF). Although aspiration is common, the clinical consequences are variable because of differences in nature of the aspirated material and individual host responses. A number of defense mechanisms normally present in the upper aerodigestive system that protect against aspiration become compromised by clinical events that occur frequently in the critical care setting, subjecting the patient to increased risk. The true incidence of aspiration has been difficult to determine in the past because of vague definitions, poor assessment monitors, and varying levels of clinical recognition. Standardization of terminology is an important step in helping to define the problem, design appropriate research studies, and develop strategies to reduce risk. Traditional clinical monitors of glucose oxidase strips and blue food coloring (BFC) should no longer be used. A modified approach to use of gastric residual volumes and identification of clinical factors that predispose to aspiration allow for risk stratification and an algorhythm approach to the management of the critically ill patient on ETF. Although the patient with confirmed aspiration should be monitored for clinical consequences and receive supportive pulmonary care, ETF may be continued when accompanied by appropriate steps to reduce risk of further aspiration. Management strategies for treating aspiration pneumonia are based on degree of diagnostic certainty, time of onset, and host factors.

In vitro release of vascular endothelial growth factor during platelet aggregation

Platelet aggregation is a cardinal feature of both vascular repair and vascular disease. During aggregation platelets release a variety of vasoactive substances; some of these promote angiogenesis, endothelial permeability, and endothelial growth, actions shared by vascular endothelial growth factor (VEGF). This study was undertaken to investigate the hypothesis that VEGF is released by aggregating platelets. We found that VEGF was secreted during the in vitro aggregation of platelet-rich plasma induced by thrombin, collagen, epinephrine, and ADP (range 23-518 pg VEGF/ml). Furthermore, serum VEGF levels were elevated compared with plasma (230 +/- 63 vs. 38 +/- 8 pg VEGF/ml), indicative of VEGF release during whole blood coagulation. Lysates of apheresed, leukocyte-poor platelet units contained significant amounts of VEGF (2.4 +/- 0.8 pg VEGF/mg protein). VEGF message and protein were also present in a megakaryocytic cell line (Dami cell). These results suggest constitutive roles for platelet VEGF in the repair of intimal vessel injury and in the altered permeability and intimal proliferation seen at sites of platelet aggregation and thrombosis.

Toll-like Receptor 1 Polymorphisms Affect Innate Immune Responses and Outcomes in Sepsis

RATIONALE Polymorphisms affecting Toll-like receptor (TLR)-mediated responses could predispose to excessive inflammation during an infection and contribute to an increased risk for poor outcomes in patients with sepsis. OBJECTIVES To identify hypermorphic polymorphisms causing elevated TLR-mediated innate immune cytokine and chemokine responses and to test whether these polymorphisms are associated with increased susceptibility to death, organ dysfunction, and infections in patients with sepsis. METHODS We screened single-nucleotide polymorphisms (SNPs) in 43 TLR-related genes to identify variants affecting TLR-mediated inflammatory responses in blood from healthy volunteers ex vivo. The SNP associated most strongly with hypermorphic responses was tested for associations with death, organ dysfunction, and type of infection in two studies: a nested case-control study in a cohort of intensive care unit patients with sepsis, and a case-control study using patients with sepsis, patients with sepsis-related acute lung injury, and healthy control subjects. MEASUREMENTS AND MAIN RESULTS The SNP demonstrating the most hypermorphic effect was the G allele of TLR1(-7202A/G) (rs5743551), which associated with elevated TLR1-mediated cytokine production (P < 2 x 10(-20)). TLR1(-7202G) marked a coding SNP that causes higher TLR1-induced NF-kappaB activation and higher cell surface TLR1 expression. In the cohort of patients with sepsis TLR1(-7202G) predicted worse organ dysfunction and death (odds ratio, 1.82; 95% confidence interval, 1.07-3.09). In the case-control study TLR1(-7202G) was associated with sepsis-related acute lung injury (odds ratio, 3.40; 95% confidence interval, 1.59-7.27). TLR1(-7202G) also associated with a higher prevalence of gram-positive cultures in both clinical studies. CONCLUSIONS Hypermorphic genetic variation in TLR1 is associated with increased susceptibility to organ dysfunction, death, and gram-positive infection in sepsis.

Vascular endothelial growth factor in pulmonary hypertension.

The best studied of the endothelial cell specific growth factors, VEGF, is present in alveolar and bronchial epithelial cells, in vascular smooth muscle cells, and in macrophages. The gene encoding VEGF is abundantly present in lung tissue and is induced by short-term and long-term hypoxia, as well as by prostacyclin, prostaglandin E2, and cyclic AMP. In the case of prostaglandin-stimulated gene upregulation protein kinase A (PKA) is involved. Suramin, an inhibitor of growth factor receptor binding inhibits the development of chronic hypoxic pulmonary hypertension in rats. Further evidence is necessary to link VEGF with pulmonary hypertensive vascular remodeling. This requires the development of antibodies directed against VEGF or against VEGF receptors and gene transfection or antisense strategies.

Genome Wide Association Identifies PPFIA1 as a Candidate Gene for Acute Lung Injury Risk Following Major Trauma

Acute Lung Injury (ALI) is a syndrome with high associated mortality characterized by severe hypoxemia and pulmonary infiltrates in patients with critical illness. We conducted the first investigation to use the genome wide association (GWA) approach to identify putative risk variants for ALI. Genome wide genotyping was performed using the Illumina Human Quad 610 BeadChip. We performed a two-stage GWA study followed by a third stage of functional characterization. In the discovery phase (Phase 1), we compared 600 European American trauma-associated ALI cases with 2266 European American population-based controls. We carried forward the top 1% of single nucleotide polymorphisms (SNPs) at p<0.01 to a replication phase (Phase 2) comprised of a nested case-control design sample of 212 trauma-associated ALI cases and 283 at-risk trauma non-ALI controls from ongoing cohort studies. SNPs that replicated at the 0.05 level in Phase 2 were subject to functional validation (Phase 3) using expression quantitative trait loci (eQTL) analyses in stimulated B-lymphoblastoid cell lines (B-LCL) in family trios. 159 SNPs from the discovery phase replicated in Phase 2, including loci with prior evidence for a role in ALI pathogenesis. Functional evaluation of these replicated SNPs revealed rs471931 on 11q13.3 to exert a cis-regulatory effect on mRNA expression in the PPFIA1 gene (p = 0.0021). PPFIA1 encodes liprin alpha, a protein involved in cell adhesion, integrin expression, and cell-matrix interactions. This study supports the feasibility of future multi-center GWA investigations of ALI risk, and identifies PPFIA1 as a potential functional candidate ALI risk gene for future research.

Food Dye Use in Enteral Feedings: A Review and a Call for a Moratorium

Pulmonary aspiration of gastric contents is common in enterally fed patients. Tinting enteral feedings with blue dye is thought to aid the early detection of aspiration in hospitalized patients. The blue-dye method is popular despite evidence that it is not sensitive. Reports of absorption of blue dye from enteral feedings in patients with sepsis and other critical illnesses are increasing. The presence of blue and green skin and urine, and serum discoloration has been linked with death. FD&C Blue No.1 and related dyes have toxic effects on mitochondria, suggesting that dye absorption is harmful. This study reviews the literature on the dye method and dye pharmacology, reports the results of a survey of current dye use, and describes 2 recent deaths associated with blue-dye absorption. We concluded that the use of blue dye in enteral feedings should be abandoned and replaced by evidence-based methods for the prevention of aspiration.

Loss-of-function thrombospondin-1 mutations in familial pulmonary hypertension

Most patients with familial pulmonary arterial hypertension (FPAH) carry mutations in the bone morphogenic protein receptor 2 gene (BMPR2). Yet carriers have only a 20% risk of disease, suggesting that other factors influence penetrance. Thrombospondin-1 (TSP1) regulates activation of TGF-β and inhibits endothelial and smooth muscle cell proliferation, pathways coincidentally altered in pulmonary arterial hypertension (PAH). To determine whether a subset of FPAH patients also have mutations in the TSP1 gene (THBS1) we resequenced the type I repeats of THBS1 encoding the TGF-β regulation and cell growth inhibition domains in 60 FPAH probands, 70 nonfamilial PAH subjects, and in large control groups. We identified THBS1 mutations in three families: a novel missense mutation in two (Asp362Asn), and an intronic mutation in a third (IVS8+255 G/A). Neither mutation was detected in population controls. Mutant 362Asn TSP1 had less than half of the ability of wild-type TSP1 to activate TGF-β. Mutant 362Asn TSP1 also lost the ability to inhibit growth of pulmonary arterial smooth muscle cells and was over threefold less effective at inhibiting endothelial cell growth. The IVS8+255 G/A mutation decreased and/or eliminated local binding of the transcription factors SP1 and MAZ but did not affect RNA splicing. These novel mutations implicate THBS1 as a modifier gene in FPAH. These THBS1 mutations have implications in the genetic evaluation of FPAH patients. However, since FPAH is rare, these data are most relevant as evidence for the importance of TSP1 in pulmonary vascular homeostasis. Further examination of THBS1 in the pathogenesis of PAH is warranted.

TGF-β activation by bone marrow-derived thrombospondin-1 causes Schistosoma - and hypoxia-induced pulmonary hypertension

Pulmonary arterial hypertension (PAH) is an obstructive disease of the precapillary pulmonary arteries. Schistosomiasis-associated PAH shares altered vascular TGF-β signalling with idiopathic, heritable and autoimmune-associated etiologies; moreover, TGF-β blockade can prevent experimental pulmonary hypertension (PH) in pre-clinical models. TGF-β is regulated at the level of activation, but how TGF-β is activated in this disease is unknown. Here we show TGF-β activation by thrombospondin-1 (TSP-1) is both required and sufficient for the development of PH in Schistosoma-exposed mice. Following Schistosoma exposure, TSP-1 levels in the lung increase, via recruitment of circulating monocytes, while TSP-1 inhibition or knockout bone marrow prevents TGF-β activation and protects against PH development. TSP-1 blockade also prevents the PH in a second model, chronic hypoxia. Lastly, the plasma concentration of TSP-1 is significantly increased in subjects with scleroderma following PAH development. Targeting TSP-1-dependent activation of TGF-β could thus be a therapeutic approach in TGF-β-dependent vascular diseases.

Expression of thrombospondins in skeletal muscle

In their FASEB Journal report, Yoshioka and colleagues (1) conclude that thrombospondins (TSP) are among a number of matrix genes that are differentially regulated in skeletal muscle of endurance athletes. The authors used an est sequence (TTGCACAGCCA) they believed was a TSP sequence as a tag ID for SAGE. That sequence actually is not any part of the coding or genomic sequences of the TSP 1-4 family (2), and cannot even be found within the companion file listed (GenBank accession AI090062) or on a BLAST search of all human est. The file AI090062 is a uterine est with similarity to a thrombospondin type I repeat superfamily gene THDS2, but not actually a TSP 1-4 family member (3). Some confusion may have arisen from similar gene codes, as the TSP1 and TSP2 genes are denoted as THBS1 and THBS2, respectively. This oversight alters the authors’ conclusions regarding differential expression of thrombospondins in muscle. It appears that members of the TSP family were, in fact, not highly expressed in muscle of either group. Furthermore, the tag for aldolase A does not map to the companion est file provided, nor can it be validated by a BLAST search. Were other tags in the SAGE software they used similarly inaccurate or mislabeled? Such results point out that interesting differential expression data obtained by methods such as SAGE and expression arrays should be validated by Northern blot or RT-PCR, neither of which was done in this study (4).

Proinflammatory Cytokines Increase Vascular Endothelial Growth Factor Expression in Alveolar Epithelial Cells

Vascular endothelial growth factor (VEGF) is an endothelial permeability mediator that is highly expressed in lung epithelium. In nonlung cells proinflammatory cytokines have been shown to increase VEGF expression, but their effects on lung epithelium remain unclear. We hypothesized that increases in alveolar epithelial cell VEGF RNA and protein expression occur after exposure to proinflammatory cytokines. We tested this using human alveolar epithelial cells (A549) stimulated with 5 proinflammatory cytokines. VEGF RNA expression was increased 1.4–2.7-fold in response to IL-1, IL-6, IL-8, TNF-α, or TGF-β over 6 hours, with TGF-β having the largest response. TNF-α increased VEGF RNA as early as 1 hour. A mix of IL-1, IL-6, and IL-8 had effects similar to IL-1. TNF-α increased protein expression as early as 4 hours and had a sustained effect at 16 hours, whereas IL-1 did not increase protein expression. Only VEGF165 was present in cultured A549 cells, yet other isoforms were seen in human lung tissue. Increased expression of VEGF in alveolar epithelial cells occurs in response to proinflammatory cytokines. Increased VEGF expression likely contributes to the pathogenesis of inflammatory lung diseases and to the angiogenic phenotype of lung cancer, a disease typically preceded by chronic inflammation.