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Introduction: Diffuse alveolar hemorrhage (DAH) is a life-threatening condition with an obscure etiology and pathogenesis. It is associated with many drugs and diseases including chemotherapy, hematopoietic stem cell transplantation, and autoimmune disorders. This retrospective study reports our experience with 23 patients who had DAH and received intravenous recombinant activated Factor VIIa (rFVIIa)., Methods: We performed a retrospective chart review of patients who received intravenous rFVIIa for DAH at a tertiary care university hospital between January 1, 2003 and May 31, 2013. We report demographics, etiology of DAH, frequency and total dose of intravenous rFVIIa, effect of rFVIIa on DAH, and morbidity and mortality., Results: Mean age was 47 ± 19 years. There were 13 men and 10 women. Nine patients had Anti-neutrophil cytoplasmic antibody (ANCA) vasculitis, two had systemic lupus erythematosus, three had Good pasture's syndrome, seven were post-bone marrow transplant, one had idiopathic thrombocytopenic purpura, and one had cryoglobulinemia. Treatment in the ICU was required for 22 patients of whom 18 were intubated and on mechanical ventilation; one patient was treated on general medical service. All patients received 35-120 mcg/kg rFVIIa every 2 h until hemostasis was achieved or treatment was judged to be inadequate. In 22/23 patients, bleeding resolved with rFVIIa therapy. The mean dose to control bleeding was 5 ± 3 mg. Eight patients died (36 %) of their underlying condition; six of them had received bone marrow transplant, while two had ANCA vasculitis. Deaths were due to multiorgan failure, sepsis, and progressive underlying disease. No overt, clinically obvious adverse thrombotic events were observed with the use of rFVIIa., Conclusion: Activated Factor VII can achieve hemostasis in patients with diffuse alveolar hemorrhage.

Cryptogenic organizing pneumonia is an idiopathic form of organizing pneumonia (also known as bronchiolitis obliterans organizing pneumonia). Because cryptogenic organizing pneumonia is considered an inflammatory disease, it characteristically responds to the broad-spectrum antiinflammatory corticosteroids, although relapse is common on discontinued use. Additionally, long-term use of corticosteroids has many side effects. In severe cases in which corticosteroids have failed, either cytotoxic therapy or macrolide therapy is used. Because of the toxicity and adverse effects of cytotoxic therapy (e.g., cyclophosphamide), this therapy option cannot be used long term in refractory cases. Macrolide therapy has been shown to be an effective antiinflammatory agent that is relatively safe when used on a long-term basis in patients with cryptogenic organizing pneumonia.

Background: Unlike Goodpasture's syndrome with diffuse alveolar hemorrhage (DAH), there are few studies examining therapy for patients with DAH associated with antineutrophil cytoplasmic antibody (ANCA)-associated small-vessel vasculitis (SVV)., Methods: We performed a retrospective review of all such patients presenting to our institution between 1995 and 2001. All patients were treated with apheresis and induction immunosuppressive therapy; namely, intravenous methylprednisolone and/or intravenous cyclophosphamide., Results: DAH resolved with apheresis in 20 of 20 patients (100%) with 6.4 (average) treatments. There were no complications of therapy. Half the patients (7 of 14) who also presented with azotemia were discharged with improved renal function., Conclusion: Patients with ANCA-related SVV and DAH benefit from prompt initiation of apheresis coupled with aggressive immunosuppressive therapy. Such therapy can be lifesaving with respect to the pulmonary component of this syndrome.

The shortage of lung donors for clinical transplantation could be significantly alleviated if lungs could be retrieved from cadavers hours after death. However, the time course of loss of lung viability after circulatory arrest and organism death remains unclear. To determine postmortem adenine nucleotide tissue levels in the lung and their relationship to lung viability, Sprague-Dawley rats were sacrificed and then ventilated with 100% oxygen (n = 50, O2) or 100% nitrogen (n = 40, N2) or left nonventilated (n = 50). Lungs from control rats (n = 20) were retrieved immediately after sacrifice. Lungs in the three study groups were retrieved at successive intervals postmortem. Adenine nucleotides (ATP, ADP, and AMP) and hypoxanthine and xanthine metabolites of adenosine were extracted from lung tissue and measured using high-performance liquid chromatography. Pulmonary parenchymal cell viability was quantified by pulmonary artery infusion of trypan blue vital dye in the contralateral lung of each animal. By 4 hr postmortem, viability was 85 +/- 1% in the O2-ventilated cadaver rat lungs, significantly higher than in the N2-ventilated (43 +/- 8%) and in the nonventilated (48 +/- 4%) lungs, where the percentage of viable cells was similar. All of the groups showed a time-dependent decrement in ATP levels and total adenine nucleotide (TAN) levels after death, but this was markedly attenuated in O2-ventilated cadaveric rat lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Inflammation in nasal and airway tissue caused by allergens, microbial infection, and air pollution are likely to be regulated by inflammatory mediators produced by airway epithelial cells. We have therefore investigated the baseline expression of a number of cytokine genes known to be important inducers and modulators of inflammation, in freshly isolated human nasal epithelium. Cells were obtained by superficial scraping of turbinate tissue, and cDNA for polymerase chain reaction (PCR) amplification was reverse-transcribed directly from lysates of 3 x 10(3) to 5 x 10(3) epithelial cells using random hexamers. Constitutive expression of relatively high levels of interleukin-8 (IL-8) mRNA but undetectable levels (< 1 mRNA copy/cell) of granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-6, IL-1, or tumor necrosis factor (TNF) mRNA were found after PCR amplification of the cDNA. IL-8 protein, but not IL-6, was identified in the nasal epithelial cells by immunocytochemistry. Infection with respiratory syncytial virus (RSV) or stimulation of nasal epithelium for 4 h with TNF or IL-1 in vitro resulted in a 4- to 10-fold increase in IL-8 mRNA expression but not in the expression of detectable levels of mRNA for the other cytokines. IL-8 was secreted by RSV-, IL-1-, and TNF-stimulated as well as unstimulated nasal epithelial cells after 6 to 20 h of culture. Neither IL-6, GM-CSF, nor TNF activity/immunoreactivity was detectable in the culture supernatants. Thus, it appears that IL-8 is a major cytokine of human nasal epithelium, constitutively expressed and readily secreted upon virus infection or stimulation with IL-1 and TNF.

Sonicates of freshly isolated Clara cells produced thromboxane B2 (TxB2), prostaglandin (PG) D2, PGE2, PGF2 alpha, hydroxyheptadecatrienoic acid (HHT), and 12-hydroxyeicosatetraenoic acid (12-HETE) as detected using high-performance liquid chromatography (HPLC). Sonicates of Clara cells cultured on collagen matrices produced the same metabolites. Rates of [3H]arachidonic acid metabolism increased in culture, but the changes were not associated with changes in cell number. Sonicates of freshly isolated tracheal cells produced mainly 12-HETE. Cyclooxygenase products were not produced consistently. Sonicates of tracheal cultures produced significant quantities of TxB2, PGD2, PGE2, PGF2 alpha, and HHT, but 12-HETE remained the major metabolite. Equivalent short-circuit current (Ieq) across cultured Clara cell epithelia was unaffected by bilateral exposure to TxB2, PGD2, PGE2, PGF2 alpha, HHT, or 12-HETE. A minor (1%) decrease in transepithelial resistance (Rt) followed exposure to PGD2. Indomethacin had no significant effect on Rt or Ieq, but exposure of indomethacin-pretreated preparations to PGE2 revealed a minor (2%) increase in Ieq. In contrast, tracheal cell epithelia exhibited significant changes in Rt and Ieq in response to PGF2 alpha, PGE2, and HHT. These results indicate that Clara cells metabolize arachidonic acid to biologically active eicosanoids, but the resulting products do not play a major role in regulation of transepithelial ion transport by this cell type.

Leukotriene B4 (LTB4) from vascular endothelium may play a key role in the genesis of atherosclerotic lesions. However, the ability of this tissue to synthesize LTB4 is controversial. To resolve this issue arachidonic acid metabolism was characterized in cultures of confluent monolayers of a rabbit aortic endothelial cell line by use of both high-pressure liquid chromatography and radioimmunoassay. Cells were grown to confluence in Dulbecco's modified Eagle's medium/Ham's F12 with 5% fetal bovine serum. Lipoxygenase activity was studied by placing the cells in Hank's balanced salt solution with 2 mumol/L indomethacin. After 30 minutes preincubation with indomethacin cells were exposed to either arachidonic acid (10 mumol/L) or arachidonic acid labeled with radioactive carbon (14C) (1 microCi; SA 58 mCi/mmol) and then stimulated with 9.5 mumol/L calcium ionophore A23187 for 55 minutes. Studies of the cyclooxygenase activity were performed without preincubating with indomethacin. Samples were prepared for high-pressure liquid chromatography by evaporation to dryness under a vacuum and resuspending in 2 ml of 1:1 methanol/water. Tritium-labeled standards were added before loading the 14C-labeled samples on the column. Radiolabeled arachidonic acid metabolites were separated by high-pressure liquid chromatography and detected by means of a dual channel flow-through radiodetector that monitors both 14C and 3H. Based on coelution with authentic standards three lipoxygenase metabolites of arachidonic acid have been identified: LTB4, 12- and 5-hydroxyeicosatetraneoic acid. Leukotriene B4 was further characterized by ultraviolet spectral analysis and inhibition studies with use of nordihydroguaiaretic acid. Quantitation was facilitated by commercially available radioimmunoassay kits. An average of 600 pg LTB4/10(6) cells was measured from separate experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Diphenylamine-2-carboxylate (DPC) decreases Cl- conductance (GCl) in epithelia and cells of several tissues, an effect which has been ascribed to blockade of conductive Cl- channels. However, one DPC derivative, flufenamic acid, is a clinically useful non-steroidal anti-inflammatory agent, the mechanism of action of which involves the blockade of arachidonic acid metabolism by cyclooxygenase. Because GCl in canine tracheal epithelium is stimulated by exogenous prostaglandins and induction of cyclooxygenase activity, we tested the hypothesis that DPC inhibits dog tracheal epithelium GCl through inhibition of cyclooxygenase. DPC inhibited the short circuit current of amiloride-pretreated tissues by 50% at 0.138 mmol/l and by more than 95% at 3 mmol/l. Isoproterenol reversed the inhibition seen at 0.1 mmol/l DPC and stimulated current above control (indomethacin-pretreated) levels. Higher concentrations of DPC diminished the stimulation of current by subsequent exposure to isoproterenol, such that there was little effect of isoproterenol in the presence of 3 mmol/l DPC. DPC, 0.1 mmol/l, also blocked stimulation of current by exogenous arachidonic acid, but not of exogenous prostaglandins PGE or PGD. The metabolism of 3H-arachidonic acid to 3H-PGD2, monitored by HPLC, was completely blocked by 0.1 mmol/l DPC. We conclude that the isoproterenol/prostaglandin reversible blockade of GCl by DPC can be attributed to inhibition of arachidonic acid metabolism.

High-performance liquid chromatography procedures were developed which separate leukotrienes (LTs), hydroxy-fatty acids (HETEs), prostaglandins (PGs), the stable metabolite of prostacyclin (6-keto-PGF1 alpha), the stable metabolite of thromboxane A2 (TXB2), 12-hydroxyheptadecatrienoic acid (HHT), and arachidonic acid (AA). Two methods employing reverse-phase columns are described. One method uses a radial compression system, the other a conventional steel column. Both systems employ methanol and buffered water as solvents. The radial compression system requires 60 min for separation of the AA metabolites, while the conventional system requires 100 min. Both methods provide good separation and recovery of 6-keto-PGF1 alpha, TXB2, PGE2, PGF2 alpha, PGD2, LTC4, LTB4, LTD4, LTE4, HHT, 15-, 12-, and 5-HETE; and AA. The 5S,12S-dihydroxy-6-trans, 8-cis, 10-trans, 14-cis-eicosatetraenoic acid (5S,12S-diHETE), a stereoisomer of LTB4, coelutes with LTB4. To determine the applicability of the methods to biologic systems, AA metabolism was studied in two models, guinea pig lung microsomes and rat alveolar macrophages. Both HPLC systems demonstrated good recovery and resolution of eicosanoids from the two biological systems. A simple evaporation technique for HPLC sample preparation, which avoids the use of chromatographic and other time-consuming methodology, is also described.

Immune deposits at the cutaneous basement membrane zone are a characteristic feature of systemic lupus erythematosus. Previous studies using immunofluorescent methods to detect complement components have provided evidence that some deposits contain immune complexes capable of activating complement. However, this important biologic property of complexes has not been detected or measured using functional assays, and it has not been determined whether immune deposits can activate complement at the basement membrane zone. In this study immune deposits in biopsies of lupus skin have been examined using direct immunofluorescence for the third component of complement (C3) to detect complement deposited in vivo. In addition, the deposits have been studied using the leukocyte attachment assay and indirect C3 binding immunofluorescence to detect and measure complement activation at the basement membrane zone in vitro. The results show that complement activation occurs at the basement membrane in some but not all lupus skin containing immunoglobulin deposits, that deposits differ quantitatively in their ability to activate complement, and that direct C3 immunofluorescence is a relatively insensitive method for detecting complement-activating complexes. The results provide functional evidence suggesting that immune deposits in some lupus skin are complement-activating complexes and potentially capable of activating complement at the basement membrane in vivo. Furthermore, the results suggest functional assays for evaluating complement-activating complexes may be valuable supplements to immunofluorescence in exploring the relationship between immune deposits and systemic and cutaneous disease.

An HPLC method for the separation and analysis of arachidonic acid and eight phospholipid classes is described: phosphatidylglycerol, phosphatidylinositol, cardiolipin, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and 2-lysophosphatidylcholine. The separation is carried out at 60 degrees C on 2 cyanopropyl columns using a gradient of acetonitrile and 5 mM sodium acetate (pH 5.0). Cyanopropyl columns require a lower proportion of water in the mobile phase to elute the more polar phospholipids than other types of columns and are thus less prone to equilibration problems. The method is highly reproducible (average coefficient of variation for each retention time less than or equal to 3.5%) and permits analysis of peaks by phosphorus content. Data obtained by analyzing lipid extracts from rat alveolar macrophages prelabeled with [G-3H]-arachidonic acid were analyzed by this HPLC method and compared to standard analysis by TLC. There was a significant correlation between the radioactivity profiles obtained with the two chromatographic methods (HPLC versus TLC) by linear regression analysis [HPLC = 0.83 (TLC) + 3.58, n = 25, r = 0.95, P less than 0.001].

Canine tracheal epithelial cells freshly isolated from mongrel dog trachea were used to study relationships between arachidonic acid metabolism and chloride ion movement. High performance liquid chromatography (HPLC) analysis of the cell incubation media after the addition of A23187 showed the presence of prostaglandin H synthase and lipoxygenase-derived metabolites. The major prostaglandin H synthase metabolite identified by HPLC, gas chromatography, and mass spectrometry was prostaglandin (PG) D2. The major lipoxygenase metabolites were leukotriene (LT) C4 and LTB4. LTB4 was identified by HPLC, UV spectroscopy, and gas chromatography. Straight phase HPLC of the methyl esters indicated only a minor formation of LTB4 isomers. LTC4 was identified by HPLC, UV spectroscopy, and conversion to LTD4 by gamma-glutamyl transpeptidase. Analysis by radioimmunoassays indicated approximately 1-2 ng of LTB4 and peptide LT formed by 10(6) cells after A23187 stimulation. The addition of ionophore A23187 caused a rapid release of arachidonic acid metabolites which was completed within 5 min of stimulation. Cl- secretion was measured in parallel studies of excised tracheas in Ussing chambers. Cl- secretion occurred at 2-3 min after the addition of ionophore, and the most rapid change occurred with the highest PGD2 concentrations. Indomethacin produced a concentration-dependent inhibition of PGD2 formation and Cl- movement. The addition of PGE2, PGD2, and PGH2 effectively stimulated Cl- secretion. LTC4 also stimulated Cl- secretion, but the stimulation was inhibited by indomethacin. These results indicate that canine tracheal epithelial cells metabolize arachidonic acid via both prostaglandin H synthase and lipoxygenase enzymes. It appears that endogenous PGD2 formation is the important variable controlling the Cl- ion movement in canine trachea.