Pseudomonas aeruginosa elastase stimulates ERK signaling pathway and enhances IL- 8 production by alveolar epithelial cells in culture

OBJECTIVE AND DESIGN: Bacterial products as well as the host airway inflammatory responses contribute to the pathogenesis of Pseudomonas infections. We sought to determine if Pseudomonas elastase (PE) induces mitogen-activated protein (MAP) kinase activity in association with interleukin-8 (IL-8) production by alveolar epithelial cells. METHODS: We utilized Western blot analysis to detect phosphorylation of signaling intermediates and ELISA was used to measure IL-8 production. RESULTS: We found that PE induces phosphorylation of the extracellular signal-regulated (ERK1/2) proteins of the MAPK pathway in A549 epithelial cells. Similar results were obtained using primary cultures of rabbit alveolar type II epithelial cells. PE also enhanced IL-8 production, which was abolished in the presence of the ERK activation inhibitor U0126. CONCLUSIONS: We conclude that PE activates the ERK1/2 arm of the MAPK pathway and that activation of this pathway results in enhanced IL-8 production. The results demonstrate that PE may augment pulmonary inflammation via cellular signaling that regulates expression of IL-8.     

Rapid change in height and body proportions of Maya American children

Maya families from Guatemala migrated to the United States in record numbers from the late 1970s to the early 1990s. Births to Maya immigrant women have created a sizable number of Maya American children. The height and sitting height of 5 to 12 years children (n = 431) were measured in 1999 and 2000. Leg length was estimated and the sitting height ratio was calculated. These data were compared with a sample of Maya children living in Guatemala measured in 1998 (n = 1,347). Maya American children are currently 11.54 cm taller and 6.83 cm longer‐legged, on average, than Maya children living in Guatemala. Consequently, the Maya Americans have a significantly lower average sitting height ratio (i.e., relatively longer legs in proportion to length of the head and trunk) than do the Maya in Guatemala. These results add support to the hypothesis that both the height and body proportions of human populations are sensitive indicators of the quality of the environment for growth. Am. J. Hum. Biol. 14:753–761, 2002. © 2002 Wiley‐Liss, Inc.     

Neutrophil defensins stimulate the release of cytokines by airway epithelial cells: modulation by dexamethasone

OBJECTIVE AND DESIGN: Neutrophils may contribute to recruiting other cells to sites of inflammation by generating chemotactic signals themselves, or by stimulating other cell types to release chemoattractants such as interleukin-8 (IL-8). Recently, we demonstrated that neutrophil-derived alpha-defensins are able to increase IL-8 expression in airway epithelial cells. In addition, it has previously been reported that neutrophil elastase-induced IL-8 synthesis was insensitive to inhibition by the glucocorticoid dexamethasone. The aim of the present study was to investigate the effect of defensins on the expression of various cytokines in cultured airway epithelial cells and to examine the effect of dexamethasone on defensin-induced cytokine synthesis in these cells. METHODS: Cultures of A549 cells and primary bronchial epithelial cells (PBEC) were stimulated with defensins either alone or in the presence of dexamethasone. Supernatants were analyzed for IL-8, ENA-78, IL-6, MCP-1 and GM-CSF by ELISA. In addition, IL-8 and ENA-78 mRNA was detected by Northern blot analysis. RESULTS: Defensins increased IL-8 expression, ENA-78, MCP-1 and GM-CSF release from A549 cells, whereas in PBEC only IL-8 and IL-6 were increased. Pre-treatment with dexamethasone significantly reduced defensin-induced IL-6, IL-8 and ENA-78 synthesis in airway epithelial cells. In addition, dexamethasone also reduced the neutrophil chemotactic activity in supernatants of these cells. CONCLUSIONS: The results from the present study indicate that defensins differentially induce cytokine secretion by A549 cells and PBEC. Glucocorticoids may interfere with the defensin-induced inflammatory process by reducing defensin-induced cytokine secretion in lung epithelial cells.     

Molecular cloning of agonistic and antagonistic monoclonal antibodies against human 4-1BB.

Previously, we prepared two different monoclonal antibodies (mAbs) against human 4-1BB (CD137): an agonistic mAb BBK-1 and an antagonistic mAb BBK-2. In this paper, we describe the molecular cloning of these two mAbs and present comparisons of their amino acid sequences. cDNAs encoding the heavy (H) and light (L) chains of the two mAbs were cloned by screening of cDNA libraries constructed from hybridomas secreting these mAbs. Comparisons of amino acid sequences of the two mAbs showed that, while the constant regions of the H and L chains were identical between the two mAbs, the variable region showed 45% identity in H chains and 48% identity in L chains. This suggests that these two mAbs recognize different epitopes of 4-1BB and may have different effects on the activity of 4-1BB.     

A randomized, double‐blind trial of the effect of glucocorticoid, antileukotriene and bβ‐agonist treatment on IL‐10 serum levels in children with asthma

BACKGROUND: Levels of an immunoregulatory and anti-inflammatory cytokine IL-10 are reduced in asthmatic airways, potentially contributing to more intense inflammation. Triamcinolone has anti-inflammatory properties and the anti-inflammatory effects of montelukast and formoterol have been discussed. OBJECTIVE: The purpose of this study was to define the effect of treatment with triamcinolone, montelukast and formoterol on the serum level of IL-10, eosinophil blood counts, eosinophil cationic response (ECP) and clinical parameters (symptom score, FEV1 and PC20H) in children with moderate asthma. METHODS: An 8-week, placebo-controlled and randomized, double-blind trial was carried out. The subjects were 91 children with moderate atopic asthma who were allergic to dust mite. Patients were randomly allocated to receive 400 microg triamcinolone (n = 19), 5 or 10 mg (according to age) montelukast (n = 18), 24 microg formoterol (n = 18) or placebo (n = 36). RESULTS: Seventy-nine children completed the study. After treatment with triamcinolone and montelukast the level of IL-10 in blood serum significantly increased, eosinophil blood counts and ECP levels significantly decreased and all clinical parameters improved; treatment with formoterol had no effect on IL-10 level, eosinophil blood counts in serum and bronchial hyper-reactivity; ECP level significantly decreased after treatment and asthma symptoms and FEV1 improved significantly. Mean IL-10 levels in serum before and after treatment with triamcinolone were 7.23 pg/mL with 95% CI, 6.74 -7.72% and 14.24 pg/mL with 95% CI, 11.6-16.88%, respectively (P < 0.001); with montelukast they were 6.59 pg/mL with 95% CI, 6.26-7.23% and 10.94 pg/mL with 95% CI, 8.24-12.65%, respectively (P < 0.002); with formoterol they were 7.06 pg/mL with 95% CI, 6.61-7.52% and 7.04 pg/mL with 95% CI, 6.15-7.93%. We found statistically significant correlations between serum level of IL-10 and serum level of ECP after treatment with triamcinolone and montelukast. CONCLUSION: This study demonstrates that one possible way by which triamcinolone and montelukast contribute to inhibition of inflammation is by increasing IL-10 levels.     

General Anesthetics Do Not Affect Release of the Neuropeptide Cholecystokinin from Isolated Rat Cortical Nerve Terminals

Background: General anesthetics inhibit evoked release of classic neurotransmitters. However, their actions on neuropeptide release in the central nervous system have not been well characterized.

Methods: The effects of representative intravenous and volatile anesthetics were studied on the release of sulfated cholecystokinin 8 (CCK8s), a representative excitatory neuropeptide, from isolated rat cerebrocortical nerve terminals (synaptosomes). Basal, elevated KCl depolarization-evoked and veratridine-evoked release of CCK8s from synaptosomes purified from rat cerebral cortex was evaluated at 35[degrees]C in the absence or presence of extracellular Ca2+. CCK8s released into the incubation medium was determined by enzyme-linked immunoassay after filtration.

Results: Elevation of extracellular KCl concentration (to 15-30 mm) or veratridine (10-20 [mu]m) stimulated Ca2+-dependent CCK8s release. Basal, elevated KCl- or veratridine-evoked CCK8s release was not affected significantly by propofol (12.5-50 [mu]m), pentobarbital (50 and 100 [mu]m), thiopental (20 [mu]m), etomidate (20 [mu]m), ketamine (20 [mu]m), isoflurane (0.6-0.8 mm), or halothane (0.6-0.8 mm).     

Comparison of gas-liquid chromatography and EMIT assay for serum valproic acid

We describe a simple direct extraction method for the gas-liquid chromatography determination of serum valproic acid. The working range for the assay is 2-180 mg/L and our within-run precision was 5.8 and 4.3% at the 40 and 90 mg/L concentrations respectively. Hemolyzed and lipemic sera as well as samples from patients with hyperbilirubinemia and from patients with decreased renal function were put through the assay and no interfering peaks were noted. Interference occurred when teflon-lined screw caps were used during the extraction step. The method was proven to be accurate by linear regression analysis of samples containing weighed-in amounts of valproic acid. The above assay was compared to an enzyme immunoassay technique (EMIT). The working range for the latter is 10-150 mg/L and the with-run precision was 10.8 and 5.9% and 90 mg/L concentration respectively. Samples were run by both the gas-liquid chromatograph and enzyme immunoassay methods and gave very similar results over the range 16-139 mg/L.