Soluble triggering receptor expressed on myeloid cell-1 is increased in patients with ventilator-associated pneumonia: a preliminary report.

RATIONALE: The diagnosis of ventilator-associated pneumonia (VAP) can be difficult. Soluble triggering receptor expressed on myeloid cell-1 (sTREM-1) has been reported to be elevated in BAL fluid from patients with VAP. OBJECTIVES: To evaluate the utility of sTREM-1 in the diagnosis of VAP in BAL fluid and the fluid collected in the expiratory trap from the ventilator, the exhaled ventilator condensate (EVC). METHODS: We prospectively collected BAL fluid and EVC from 23 patients clinically suspected of having VAP. A sensitive enzyme-linked immunosorbent assay was developed to measure sTREM-1. The results derived from this assay were confirmed using an immunoblot technique. The presence of VAP was clinically determined using a modified clinical pulmonary infection score of > 6. RESULTS: VAP was diagnosed in 14 of 23 patients. sTREM-1 was detected in the EVC from 11 of 14 subjects with VAP, but from only 1 of 9 subjects without VAP, and was significantly higher in the pneumonia patients and when expressed as picograms per milliliter or picograms per microgram protein (p = 0.005, both comparisons). In contrast, sTREM-1 was detected in the BAL fluid of all 14 VAP subjects but also in 8 of 9 subjects with no pneumonia, and did not differ in the VAP subjects compared to the nonpneumonia subjects when expressed as picrograms per milliliter or picograms per microgram protein (p > 0.05 both comparisons). CONCLUSION: sTREM-1 is detectable in EVC and may be useful in establishing or excluding the diagnosis of VAP.

Doxycycline decreases monocyte chemoattractant protein-1 in human lung epithelial cells.

Certain antibiotics possess anti-inflammatory properties and could potentially be used to treat inflammatory lung diseases associated with an influx of monocytes such as panbronchiolitis, asthma, cystic fibrosis, and bronchitis. Doxycycline is reported to possess anti-inflammatory effects. Monocyte chemoattractant protein-1 (MCP-1) is a major inflammatory cytokine and a powerful chemoattractant for monocytes. The authors hypothesized that doxycycline exerts its anti-inflammatory effects, in part, by reducing MCP-1 production. To test this hypothesis, A549 human lung epithelial cells were stimulated with cytomix in the presence or absence of doxycycline. In stimulated cells doxycycline decreased MCP-1 production by 95% and in monocyte chemotaxis assays migration decreased by 55%. However, doxycycline did decrease expression of MCP-1 mRNA and did not effect its stability. These data demonstrate that doxycycline modulates MCP-1 production and suggest that doxycycline may provide a new anti-inflammatory therapy for chronic lung diseases.

Doxycycline modulates nitric oxide production in murine lung epithelial cells.

Many effective therapeutic agents exhibit effects that are different from their intended primary mode of action. Antibiotics such as doxycycline and erythromycin A are no exception. They also display anti-inflammatory activity. Using LA4 murine lung alveolar epithelial cells, effects of doxycycline and erythromycin A on inducible NO synthase (iNOS) NO production as well as iNOS protein and mRNA production were investigated. Induction of iNOS was accomplished by treatment with cytomix (TNF-alpha, IL-1beta, and IFN-gamma each at 5 ng/ml). Production of NO or iNOS was not detected in controls with or without erythromycin A. In the presence of cytomix, erythromycin A did not decrease NO, nitrite, iNOS protein, or mRNA production. In contrast, doxycycline caused a dose-dependent decrease in NO, nitrite, iNOS protein, and mRNA production in cytomix-treated cells. Doxycycline at 30 mug/ml produced a 90% decrease in nitrite and NO production and a 52% decrease in iNOS mRNA transcription compared with cytomix treatment alone. Actinomycin D treatment suggests that doxycycline decreases stability of iNOS mRNA in cytomix-treated cells. To determine a mechanism for the decrease in iNOS expression, NF-kappaB and AP-1 transcription regulatory systems and p38 MAPK were examined. Doxycycline treatment gave no statistically significant change in NF-kappaB activation but did decrease p38 MAPK protein in cytomix-treated cells by 50%, suggesting that p38 MAPK may be responsible for stabilization of iNOS mRNA. These results demonstrate that doxycycline decreases NO production from iNOS by destabilization of iNOS mRNA via decreased expression of p38 MAPK.

Heat decreases formoterol delivery.

STUDY OBJECTIVES: Based on anecdotal reports of formoterol aggregating in mailboxes in the summer in Arizona, we examined the effect of heat on formoterol as well as on drug delivery. DESIGN: Formoterol capsules in original blister packaging were heated to 40 to 70 degrees C (104 to 158 degrees F) for 3 h and at 70 degrees C (158 degrees F) for 15 to 180 min. Capsules were removed from packaging, and a vacuum setup was used to dispense the formoterol into a filter using the device provided by the manufacturer. The weights of the capsule predispensation and postdispensation were measured to calculate drug delivery. Measurements were compared to those of capsules not exposed to heat. For comparison, tiotropium and a combination of fluticasone propionate and salmeterol (Advair; GlaxoSmithKline; Research Triangle Park, NC) were similarly tested. RESULTS: Visual inspection of the heated capsules revealed gross distortion as well as visible clumping of formoterol at the higher temperatures. The mean (+/- SEM) change in the weights of capsules that underwent heating were significantly less than those obtained from capsules that had not been heated (mean change after heating for 3 h at 70 degrees C, 2.3 +/- 0.7 vs 24.7 +/- 0.6 mg, respectively; p < 0.001), indicating decreased formoterol delivery. Heat produced a dose-responsive and time-responsive decrease in formoterol delivery. One of six capsules that were subjected to temperatures as low as 40 degrees C (104 degrees F) for 3 h had decreased delivery, and three of six capsules subjected to a temperature of 70 degrees C (158 degrees F) for times as short as 30 min decreased delivery. In contrast, neither tiotropium nor fluticasone propionate/salmeterol delivery was decreased by heating for up to 3 h at 70 degrees C (158 degrees F). Thermometers placed in mailboxes or in car windows in mid-summer in Arizona (approximate outside temperature, 110 degrees F [43 degrees C]) exceeded 70 degrees C (158 degrees F). CONCLUSIONS: These data demonstrate that the exposure of formoterol to heat decreases drug delivery and that caution should be used when mailing, transporting or storing formoterol.

Serine protease inhibitors modulate smoke-induced chemokine release from human lung fibroblasts.

Smoking is associated with lung inflammation and a protease-antiprotease imbalance. We previously reported that cigarette smoke extract (CSE) stimulates human lung fibroblasts to release chemotactic cytokines. We hypothesized that serine protease inhibitors might modulate lung fibroblast release of chemotactic cytokines in response to CSE. To test this hypothesis, serine protease inhibitors (FK706, alpha1-antitrypsin, methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone, or Nalpha-p-tosyl-L-lysine chloromethyl ketone) were evaluated for their capacity to attenuate the release of neutrophil chemotactic activity (NCA) and monocyte chemotactic activity (MCA) from human fetal lung fibroblasts by the blind-well chemotactic chamber. Metalloproteinases and cysteine proteinases were not examined in this study. Similarly, the release and gene expression of chemokines and nuclear factor-kappaB (NF-kappaB) activation were measured by means of enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction. Release of NCA, MCA, chemotactic chemokines including interleukin-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, and granulocyte-macrophage colony-stimulating factor, and the expression of interleukin-8 and monocyte chemoattractant protein-1 mRNA were attenuated by FK706. Furthermore, FK706 suppressed NF-kappaB activation. These data suggest that serine protease inhibitors attenuate the CSE-induced release of NCA and MCA from human fetal lung fibroblasts and that the inhibitory action of antiproteases might depend on NF-kappaB signaling pathway.

Serine protease inhibitors modulate chemotactic cytokine production by human lung fibroblasts in vitro.

Chemotactic chemokines can be released from lung fibroblasts in response to interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. An imbalance between proteases and antiproteases has been observed at inflammatory sites, and, therefore, protease inhibitors might modulate fibroblast release of chemotactic cytokines. To test this hypothesis, serine protease inhibitors (FK-706, alpha(1)-antitrypsin, or N(alpha)-p-tosyl-L-lysine chloromethyl ketone) were evaluated for their capacity to attenuate the release of neutrophil chemotactic activity (NCA) or monocyte chemotactic activity (MCA) from human fetal lung fibroblasts (HFL-1). Similarly, the release of the chemoattractants IL-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, macrophage colony-stimulating factor, and granulocyte/macrophage colony-stimulating factor, from HFL-1, were evaluated in response to IL-1beta and TNF-alpha. NCA, MCA, and chemotactic cytokines were attenuated by FK-706. However, matrix metalloproteinase inhibitors were without effect, and cysteine protease inhibitors only slightly attenuated chemotactic or cytokine release. These data suggest that IL-1beta and TNF-alpha may stimulate lung fibroblasts to release NCA and MCA by a protease-dependent mechanism and that serine protease inhibitors may attenuate the release.

Cigarette smoke decreases inducible nitric oxide synthase in lung epithelial cells.

Cigarette smoking has been associated with decreased exhaled nitric oxide (NO). To investigate the mechanism of this decrease, the effects of a cigarette smoke extract were evaluated a murine lung epithelial cell line (LA-4), a human lung epithelial cell line (A549), and primary cultures of human lung epithelial cells induced to produce NO by cytokines. NO production was evaluated by measuring nitrite, a stable end product of NO, in cell culture supernatant fluids. Cigarette smoke extract caused a reduction in the cytokine-induced nitrite concentrations in the culture supernatant fluids from all 3 cell types (P < .01, all comparisons). To further investigate these observations, immunohistochemistry demonstrated a decrease in cytokine-induced inducible NO synthase (iNOS) protein expression and iNOS mRNA after cigarette smoke extract exposure in LA-4 cells. However, iNOS mRNA half-life was not altered by the smoke extract, suggesting that the smoke extract decreased NO by decreasing iNOS mRNA transcription. These findings demonstrate that cigarette smoke extract decreases iNOS expression and NO production from lung epithelial cells.

Peroxynitrite enhances interleukin-10 reduction in the release of neutrophil chemotactic activity.

Peroxynitrite, formed by nitric oxide and superoxide, has been shown to nitrate and reduce the function of proinflammatory proteins such as interleukin (IL)-8, monocyte chemoattractant protein-1, and eotaxin, but in contrast, to enhance the function of the anti-inflammatory cytokine IL-10 in reducing IL-1 release from blood monocytes. However, the effect of nitrated IL-10 on release of proinflammatory cytokines from lung epithelial cells is unknown. We hypothesized that peroxynitrite would enhance the capacity of human IL-10 to reduce inflammatory mediators released by epithelial cells. To test this hypothesis, recombinant human IL-10 was evaluated for its capacity to attenuate the release of neutrophil chemotactic activity and IL-8 from a human epithelial cell line in response to IL-1 beta and tumor necrosis factor-alpha. Neutrophil chemotactic activity and IL-8 in lung epithelial culture supernatant fluids were significantly lower after culture with nitrated human IL-10 compared with non-nitrated human IL-10 controls (P < 0.05). Consistent with these results, nitrated human IL-10 attenuated IL-8 mRNA expression more than non-nitrated human IL-10 controls (P < 0.05). These data demonstrate that peroxynitrite exposed human IL-10 has enhanced anti-inflammatory activity and suggest that nitration may play a critical role in the regulation of inflammation within the lower respiratory tract.

Enhanced activity of human IL-10 after nitration in reducing human IL-1 production by stimulated peripheral blood mononuclear cells.

Nitric oxide and superoxide form the unstable compound, peroxynitrite, which can nitrate proteins and compromise function of proinflammatory cytokines at sites of inflammation. Reduced function of proinflammatory proteins such as IL-8, macrophage inflammatory protein-1alpha, and eotaxin suggest an anti-inflammatory effect of nitration. The effects of nitration on anti-inflammatory cytokines such as IL-10 are unknown. We hypothesized that peroxynitrite would modify the function of anti-inflammatory cytokines like IL-10. To test this hypothesis, the capacity of recombinant human IL-10 to inhibit production of human IL-1beta (IL-1) from LPS-stimulated human PBMC was evaluated. Human IL-10 was nitrated by incubation with peroxynitrite or by incubation with 3-morpholinosydnonimine, a peroxynitrite generator, for 2 h and then incubated with LPS-stimulated PBMC for 6 h, and IL-1 was measured in the culture supernatant fluids. Human IL-1 production was significantly lower in the peroxynitrite- or 3-morpholinosydnonimine-nitrated IL-10 group than in the IL-10 controls (p < 0.05, all comparisons). This finding demonstrates that although peroxynitrite inhibits proinflammatory cytokines, it may augment anti-inflammatory cytokines and further point to an important role for peroxynitrite in the regulation of inflammation.

Molecular characterization and analysis of the biosynthetic gene cluster for the azoxy antibiotic valanimycin.

Streptomyces viridifaciens MG456-hF10 produces the antibiotic valanimycin, a naturally occurring azoxy compound. Valanimycin is known to be derived from valine and serine with the intermediacy of isobutylamine and isobutylhydroxylamine, but little is known about the stages in the pathway leading to the formation of the azoxy group. In previous studies, a cosmid containing S. viridifaciens DNA was isolated that conferred valanimycin production upon Strepto-myces lividans TK24. Subcloning of DNA from the valanimycin-producing cosmid has led to the identi-fication of a 22 kb segment of DNA sufficient to allow valanimycin production in S. lividans TK24. Sequencing of this DNA segment and the surrounding DNA revealed the presence of 20 genes. Gene disruption experiments defined the boundaries of the valanimycin gene cluster, which appears to contain 14 genes. The cluster includes an amino acid decar-boxylase gene (vlmD), a valanimycin resistance gene (vlmF ), at least two regulatory genes (vlmE, vlmI ), two genes encoding a flavin monooxygenase (vlmH, vlmR), a seryl tRNA synthetase gene (vlmL ) and seven genes of unknown function. Overproduction and characterization of VlmD demonstrated that it catalyses the decarboxylation of l-valine. An unusual feature of the valanimycin gene cluster is that four genes involved in branched amino acid biosynthesis are located near its 5' end.