The effect of enantiomers of beta-agonists on myofibroblast-derived vascular endothelial growth factor and other matrix components in the presence of dust-mite extract.

Beta(2)-adrenergic receptor agonists have been shown to modulate airway epithelial cell and smooth muscle release of cytokines and growth factors transforming growth factor (TGF) beta, associated with remodeling, is known to up-regulate the synthesis of vascular endothelial growth factor (VEGF) and stimulate differentiation of fibroblasts to the myofibroblast phenotype. VEGF and fibronectin can promote angiogenesis and (S)-albuterol can induce VEGF secretion from normal human lung fibroblasts (NHLF). We hypothesize that (S)-albuterol could stimulate myofibroblast secretion and expression of VEGF and fibronectin in the presence of Dermatophagoides pteronyssinus extract. Cultured NHLFs were stimulated with IL-1beta, TGF-beta, D. pteronyssinus, and treated with (R)- and (S)-enantiomers of albuterol. VEGF and fibronectin and basic fibroblast growth factor (bFGF) were measured by ELISA and mRNA. VEGF secretion by fibroblasts was twofold higher with 10(-7) M of (R) relative to (S) (p < 0.05). Myofibroblast secretion of VEGF was increased twofold over fibroblasts, but there was no difference between enantiomers. (S)-albuterol at 10(-8)-10(-4) M caused an increase in VEGF mRNA that paralleled VEGF secretion relative to 10(-8)-10(-4) M. Fibronection secretion by myofibroblasts but not fibroblasts was increased by 10(-5) M of (S) relative to (R) in the presence of recombinant interleukin 1 (rhIL-1)beta and D. pteronyssinus (S)-albuterol at 10(-6) M increased bFGF. The 10(-6) M of (S)-albuterol, but not (R)-albuterol, may promote angiogenesis. Increased fibronectin or bFGF by (S)-albuterol could enhance matrix deposition and remodeling in a subset of asthmatic patients.

Vascular endothelial growth factor is increased by human pulmonary cells stimulated with Dermatophagoides sp. extract.

Airway remodeling may lead to increased secretion of TGF-beta. TGF-beta is known to activate fibroblasts and to stimulate the secretion of vascular endothelial growth factor (VEGF). Soluble and cell-associated VEGF have been identified in subjects with chronic severe asthma. Dermatophagoides sp. has been shown to contribute to the pathogenesis of asthma. The aim of this study was to determine if the Dermatophagoides sp. extract can stimulate confluent A549 (cA549) to express soluble or cell-associated VEGF and to secrete other factors that stimulate normal human lung fibroblasts (NHLFs) to secrete VEGF. Immunocytochemistry for cell-associated VEGF (pg/mL) was performed on cA549 stimulated with 300, 600, and 1000 AU/mL of dialyzed Dermatophagoides sp. extract for 24 hours with serum-free media (SFM). The subsequent conditioned media (CM) was transferred to NHLF for 24 hours (CM-NHLF). VEGF in CM, CM-NHLF, and control media (CTLM; extract without cA549) were measured by ELISA and normalized to cell density as measured by absorbance of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide at A550 nm (VEGF pg/mL:A550). Parametric data were analyzed by t-test or ANOVA at alpha = 0.05. Dermatophagoides sp. extract increased normalized VEGF secretion by cA549. Immunochemical VEGF expression by cA549 was increased qualitatively by Dermatophagoides sp. extract. The 1000 CM-NHLF showed increased normalized VEGF relative to CTLM. Dermatophagoides sp. stimulates cA549 to increase soluble and immunochemical VEGF and to secrete mediators that stimulate NHLF to increase secretion of VEGF. This suggests that inhalation of dust-mite allergen may stimulate airway cells to increase VEGF secretion, potentially contributing to edema in airway remodeling.

Dermatophagoides extract-treated confluent type II epithelial cells (cA549) and human lung mesenchymal cell growth.

BACKGROUND: Chronic severe persistent asthma is associated with damaged epithelial cells with discontinuous tight junctions that contribute to dysregulated fibroblast and endothelial cell (mesenchymal) growth. Dermatophagoides species-derived proteases have been shown to cause damage to epithelial cell tight junctions. OBJECTIVE: To determine whether Dermatophagoides species can stimulate confluent A549 (cA549), a cell type with discontinuous tight junctions that approximate differentiated type II cells, to undergo altered growth and secrete putative soluble factors that affect the growth of human lung fibroblasts and microvascular endothelial cells. METHODS: Dialyzed Dermatophagoides pteronyssinus or Dermatophagoides farinae extracts (0, 300, 600, and 1000 AU/mL) were cultured with and without cA549 in serum-free media for 24 hours. After changes in cA549 growth were recorded, conditioned media from extracts with cA549 (CM) and without cA549 (control media [CTLM]) were transferred to fibroblasts and endothelial cells for 24 hours. Fibroblast and endothelial cell growth responses to CM and CTLM were observed and measured. RESULTS: All conditions showed greater than 95% cell viability. Confluent A549 showed dose-dependent growth changes characterized by increased aggregation when incubated with 300, 600, and 1000 AU/mL of D pteronyssinus in serum-free media relative to control. The CM, but not the CTLM, induced dose-dependent aggregation by fibroblasts and endothelial cells. Fibroblasts also showed decreased adhesion when incubated with CM. Dermatophagoides farinae-treated cA549 showed similar but weaker results. The use of serum, boiled CM, or boiled extract inhibited these findings. CONCLUSIONS: Dialyzed Dermatophagoides species extracts altered cA549 growth and stimulated the secretion of factors that dysregulate mesenchymal cell growth in vitro.

Detection of bacteria in WBC-reduced PLT concentrates using percent oxygen as a marker for bacteria growth.

BACKGROUND: The risk of receiving a PLT concentrate (PC) contaminated with bacteria may be 1000-fold greater than that of pathogenic viral transmission, yet surveillance for this risk is not generally practiced. A novel bacteria detection system (BDS) that overcomes the limitations of current systems is described. The BDS monitors percent oxygen (%O2) in air above aliquots of PCs that have been filtered to remove the confounding effect of respiring PLTs and residual WBCs. STUDY DESIGN AND METHODS: One-day-old WBC-reduced whole-blood-derived PCs (WBPCs) were inoculated with bacteria at 100 to 500 CFU per mL. After 30 minutes, 2- to 3-mL aliquots were processed through a PLT-reducing filter into a sample pouch containing sodium polyanethol sulfonate and entrained air. After incubation at 35 degrees C for at least 24 hours, the %O2 was measured within the pouch. Noninoculated WBC-reduced WBPCs (n = 155), confirmed free of bacteria by routine culture, were tested in a like manner. Results from the latter group of WBC-reduced WBPCs were used to distinguish contaminated from noncontaminated units. RESULTS: After a 24-hour incubation at 35 degrees C, 195 (96.5%) of the 202 sample pouches obtained from inoculated units were detected by the BDS. After an additional 6 hours at room temperature, those that remained and were tested were found positive. None of the noninoculated controls produced a positive reading. CONCLUSION: The BDS is easy to use and provides good levels of sensitivity and specificity.

A review of leukofiltration in cardiac surgery: the time course of reperfusion injury may facilitate study design of anti-inflammatory effects.

The systemic inflammatory response syndrome (SIRS) is a well-recognized phenomenon attending cardiopulmonary bypass (CPB) surgery. SIRS leads to costly complications and several strategies intended to ameliorate the symptoms have been studied, including leukocyte reduction using filtration. Although the body of work suggests that leukoreduction attenuates SIRS, discrepancies remain within the literature. The recent literature is reviewed, highlighting the areas where concordance is lacking. Investigations into many promising device-related technologies are often deterred by the high costs of clinical trials. Adding to costs is the fact that clinical end points generally require large sample sizes. An understanding, however, of the pathogenesis of reperfusion injury can guide the investigator to choose physiologic response measures that correlate well with clinical outcome, but feature low inherent variability, allowing for clinical trials with smaller sample sizes. With this goal in mind, a model for the pathogenesis of reperfusion injury is described. Using a model of reperfusion injury as underpinnings for the design of prospective pilot studies, we show that salvaged blood reinfused following CPB elicits time-dependent effects on pulmonary function as predicted by the model. Data are illustrative of principles that could expand the scope of clinical investigations designed to validate the use of physiologic response measures as correlates of clinical outcome. Such investigations would target surrogate markers of clinical outcome, measured at clinically relevant times. Once validated, these surrogate markers would, thereafter, become economical screening tools for clinical studies of device-related or pharmacological anti- inflammatory interventions.

A review of leukofiltration therapy for decreasing the morbidity associated with cardiopulmonary bypass and acute inflammatory bowel disease.

Complications of cardiopulmonary bypass (CPB) and acute inflammatory bowel disease (IBD) are associated with increased morbidity and cost. During reperfusion post-CPB, activated neutrophils adhere to microvascular endothelial cells mediated by cell adhesion molecules (CAMs) and cytokines/chemokines with subsequent myocardial damage caused by activated neutrophil-derived oxidants and enzymes. Leukofiltration was shown to reduce myocardial reperfusion injury and improve gas exchange as suggested by improvements in surrogate markers of inflammation and clinical end points. In acute IBD, characterized by rectal bleeding and protracted hospital stays, circulating neutrophils emigrate to the inflamed colon and adhere to microvascular endothelial cells by CAMs. Multiple treatments with leukofiltration in IBD were shown to induce long-term remission of acute IBD. Hence, leukofiltration may reduce reperfusion injury and rectal bleeding in CPB and IBD, respectively, and therefore decrease the morbidity and cost associated with these diseases.