Inhibition of TNF-alpha processing and TACE-mediated ectodomain shedding by ethanol.

Alcohol (EtOH) is a well-documented immunosuppressant. Acute EtOH-induced immunosuppression is partially due to suppression of tumor necrosis factor alpha (TNF-alpha) secretion. We investigated the mechanism of acute EtOH-induced TNF-alpha suppression in two monocytic cell lines, Mono Mac 6 and DRM. EtOH inhibited TNF-alpha secretion in a dose-dependent manner. However, TNF-alpha transcription was not affected by EtOH. Enzyme-linked immunosorbent assay and confocal microscopy showed that EtOH treatment increased cell-associated TNF-alpha. Ectodomain shedding of TNF-alpha from the cell surface is mediated by TNF-alpha converting enzyme (TACE). In contrast with TNF-alpha, EtOH did not inhibit interleukin-8 (IL-8) secretion, which does not require shedding. Furthermore, TNF p75 receptor shedding, a biomarker for TACE activity, was inhibited by EtOH in both cell lines. EtOH also inhibited TNF p75 receptor shedding in TACE-reconstituted fibroblasts, suggesting that EtOH inhibits the shedding process. These data show that acute EtOH exposure can posttranscriptionally suppress TNF-alpha production, resulting in specific defects in immune defense.

Use of transient CD4 lymphocyte depletion to prolong transgene expression of E1-deleted adenoviral vectors.

E1-deleted adenoviral vectors are increasingly being utilized for in vivo gene transfer. The potential use of these vectors is limited by transient expression of the transgene and a markedly reduced rate of transduction following readministration, presumably due to a host immune response to the vector. We hypothesized that CD4+ lymphocytes are necessary to generate an immune response to these vectors and that administration of a depleting anti-CD4 antibody (GK1.5) might prolong transgene expression in vivo. We found that pretreatment of mice with a single injection (transient depletion) or weekly injections of GK1.5 (persistent depletion), markedly prolonged expression of an adenovirus-encoded tumor necrosis factor (TNF) inhibitor or luciferase gene compared to controls. Moreover, mice treated with GK1.5 showed no antiadenoviral antibody response to repeat administration of the vector and a second adenoviral transgene could be expressed in these animals. However, control mice developed a significant neutralizing antibody response that prevented transgene expression with administration of a second adenovirus. These findings demonstrate that manipulation of the host immune response may expand potential applications of gene transfer utilizing adenoviral vectors.

Regulation of tumor necrosis factor cytotoxicity by calcineurin.

Cyclosporin (CsA) inhibits mitochondrial death signaling and opposes tumor necrosis factor (TNF)-induced apoptosis in vitro. However, CsA is also a potent inhibitor of calcineurin, a phosphatase that may participate in cell death. Therefore, we tested the hypothesis that calcineurin regulates TNF cytotoxicity in rat hepatoma cells (FTO2B). TNF-treated FTO2B cells appeared apoptotic by DNA fragmentation, nuclear condensation, annexin V binding, and caspase activation. We studied two calcineurin inhibitors, CsA and FK506, and found that each potently inhibited TNF cytotoxicity. Western blot demonstrated calcineurin in FTO2B homogenates. In a model of mitochondrial permeability transition (MPT), we found that CsA prevented MPT and cytochrome c release, while FK506 inhibited neither. In summary, we present evidence that calcineurin participates in an apoptotic death pathway activated by TNF. CsA may oppose programmed cell death by inhibiting calcineurin activity and/or inhibiting mitochondrial signaling.

Comparison of Xe-133 washout and single-breath imaging for the detection of ventilation abnormalities.

The Xe-133 ventilation studies of 115 patients were analyzed to determine the relative abilities of the single-breath and washout phases to detect regional ventilation abnormalities. All Xe-133 images were obtained in the posterior projection before 6-view perfusion studies with Tc-99m human albumin microspheres. There were 275 regions with matching V-P abnormalities in the patients. The washout portion of the study detected 258 of these regions (94%) and the single breath detected 175 (64%) (p less than 0.01). The discrepancies were confined to regions with nonsegmental perfusion defects, where the single breath detected 139 matches and the washout 216. The discrimination ratio between normal areas and areas of obstructive lung disease improved from 2 to 1 after 1 min washout to 30 to 1 after 5 min. The late phases of Xe-133 washout are useful in detecting ventilation abnormalities, especially those associated with nonsegmental perfusion defects.

Postpartum acute myocardial infarction: a rare occurrence of uncertain etiology.

Two women who had an acute myocardial infarction during the early postpartum period are described, and the findings in nine other women previously reported on are summarized. Attention is called to the atypical nature of ischemic heart disease and myocardial infarction in these patients when compared with myocardial infarction during pregnancy and with ischemic heart disease in men. Postpartum infarcts most often occur in women in their 20s during their first pregnancy, frequently a pregnancy complicated by the preeclampsia-eclampsia syndrome, and are associated with a high mortality rate. In one of our patients and one patient previously reported on, the results of coronary arteriography were normal, suggesting that coronary arterial thromboembolism or spasm was a cause of the infarction. Because postpartum myocardial infarction may be unrelated to atherosclerotic narrowing, detailed angiographic studies in such patients appear warranted. These cases indicate the variety and complexity of ischemic heart disease in women.

Frequency and prognostic significance of pericardial effusion in primary pulmonary hypertension. PPH Study Group. Primary pulmonary hypertension.

Pericardial effusion was noted in 43 of 79 patients (54%) with severe primary pulmonary hypertension. Larger effusion was associated with hemodynamic and echocardiographic evidence of right heart failure, impaired exercise tolerance, and a poor 1-year prognosis.

Serine/threonine phosphorylation in cellular signaling for alveolar macrophage phagocytic response to endotoxin.

Protein serine/threonine (ser/thr) phosphorylation is an early signaling event in macrophage activation. We investigated the changes in stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) activity and effects of phosphatase inhibition on alveolar macrophage (AM) function in rats challenged with intratracheal endotoxin. Animals were sacrificed 90 min post intratracheal lipopolysaccharide (LPS, 100 microg/rat) challenge. AMs were incubated with or without phosphatase inhibitors at 37 degrees C for 30 min. Phagocytosis, CD18 expression, SAPK/JNK and phosphatase activities of AMs were determined. LPS challenge activated SAPK/JNK activity and enhanced phagocytosis of AMs without altering phosphatase activity in these cells. Inhibition of phosphatase 1 and 2A activity with okadaic acid and calyculin A exerted a bi-phasic effect on AM phagocytic function. Okadaic acid at a concentration of 1 microM increased the mean channel fluorescence intensity (MCF) and the percentage of cells engaged in phagocytosis (percent phagocytosis) in AMs from saline-treated rats. This inhibitor at concentrations of 0.5 and 1 microM enhanced both the MCF and percent phagocytosis of AMs from LPS-challenged rats. Calyculin A at a concentration of 10 nM increased the MCF phagocytosis of AMs from LPS-challenged rats. At higher concentrations (20 and 30 nM), calyculin A showed a suppression on both the MCF and percent phagocytosis of AMs in both saline and LPS groups. AM CD18 expression was not altered following LPS challenge. Phosphatase inhibitors at doses that enhanced AM phagocytosis showed either no effect (okadaic acid) or inhibition (calyculin A) of AM CD18 expression. These results suggest that ser/thr phosphorylation and dephosphorylation participate in mediating the phagocytic response of AMs to LPS.

Modulation of the lung host response by granulocyte colony-stimulating factor in rats challenged with intrapulmonary endotoxin.

The effects of granulocyte colony-stimulating factor (G-CSF) on the functional activities of circulating and lung-recruited neutrophils (PMNs) and alveolar macrophages (AMs) were studied in rats to further elucidate the mechanisms underlying G-CSF-enhanced pulmonary host defense. Animals received G-CSF or vehicle twice a day for 2 days, followed by an intratracheal challenge with endotoxin or saline. G-CSF up-regulated CD11b/c expression and mean channel fluorescence intensity of phagocytosis in circulating PMNs. G-CSF also enhanced phagocytic activities, reflected by both the percentage of phagocytosis and mean channel fluorescence intensity in lung-recruited PMNs and AMs in intratracheal endotoxin-challenged rats. The endotoxin-induced increase in pulmonary production of tumor necrosis factor-alpha and cytokine-induced neutrophil chemoattractant was not affected by G-CSF pretreatment. These data demonstrate that G-CSF-enhanced pulmonary recruitment of PMNs is primarily based on the effects of G-CSF on the PMNs themselves, rather than the generation of certain chemotactic stimuli, i.e., cytokine-induced neutrophil chemoattractant and tumor necrosis factor-alpha. The enhanced phagocytic activities of lung-recruited PMNs and AMs also augment pulmonary host defenses in G-CSF-pretreated animals.

Cardiac augmentation by phasic high intrathoracic pressure support in man.

Left ventricular performance can be significantly influenced by changes in intrathoracic pressure. In man, sustained increases in intrathoracic pressure unload the left ventricle, but since venous return decreases, increased intrathoracic pressure is associated with a decreased cardiac output. In a canine model of acute ventricular failure, it has been shown that phasic increases in intrathoracic pressure, which do not decrease venous return, improve steady-state cardiac output. We thus studied the cardiovascular effects of phasic high intrathoracic pressure support (PHIPS) in seven patients with shock in our intensive care unit whose condition was not responsive to conventional types of therapy. The PHIPS was generated by abdominal and chest wall binding during positive-pressure ventilation. As compared to the state before PHIPS, the PHIPS was associated with an increase in esophageal pressure (6.6 +/- 1.1 mm Hg; p less than 0.01) and in mean arterial pressure (43.0 +/- 6.1 to 51.0 +/- 7.7 mm Hg; p less than 0.01) while not changing arterial pressure relative to esophageal pressure. Cardiac output also increased from 3.6 +/- 0.5 to 4.2 +/- 0.6 L/min (p less than 0.05), while left ventricular filling pressures remained constant. In one subject a gated cardiac blood pool scan demonstrated a PHIPS-associated increase in ejection fraction and decreased end-diastolic volume. These results are consistent with the hypothesis that PHIPS, by increasing intrathoracic pressure, augments left ventricular performance by reducing left ventricular afterload. This appears to be a promising area for future research.

The effects of acute ethanol ingestion on pulmonary diffusing capacity.

Single breath diffusing capacities for carbon monoxide (DLco) were measured in 12 normal supine subjects before and after ingestion of 15 to 30 ml of 95 percent ethanol (ETOH) to determine if alcohol could acutely change the DLco. Both DLco and specific DLco (DLco/alveolar volume) were significantly decreased 90 minutes after the ingestion of alcohol. This change may be due to a direct effect of alcohol on the alveolar capillary interface by possibly interfering with a carbon monoxide carrier molecule. Another plausible explanation for the reduced diffusing capacity after ingestion of alcohol may be redistribution of blood from the lung to the periphery secondary to the hemodynamic effects of alcohol.

Severe viral pneumonia in young adults.

Three patients with primary group-A influenzal pneumonia had diffuse pulmonary infiltrates, arterial oxygen tensions (PaO2) less than 50 mm Hg while breathing oxygen at 1 atm (fractional concentration of oxygen in the inspired gas (FIo2) equals 1.0), and right-to-left pulmonary shunts greater than 45 percent of total pulmonary blood flow. At an FIo2 of 1.0, end-expiratory pressure (EEP) was added in increments of 2 to 5 cm H2O every 30 to 60 minutes until the PaO2 was above 200 mm Hg and right-to-left shunting had fallen to less than 25 percent. The FIo2 was then lowered to 0.5. Using this systematic approach, all three patients required an FIo2 of 1.0 for less than 12 hours, minimizing the risk of oxygen toxicity. Two of the three patients did not require mechanical ventilation and breathed spontaneously while on continuous positive airway pressure (CPAP), and one of them tolerated an EEP of 31 cm H2O. Two patients survived, and one died of a neurologic complication of cardiopulmonary arrest, despite clearing on the chest x-ray film and improved gas exchange. Therapy with CPAP can be safely used in adults and has practical as well as theoretic benefits over continuous positive-pressure ventilation.

Response of patients with chronic obstructive lung disease to the regular administration of nebulized isoproterenol. A double-blind crossover study.

The effect of the regular use of neublized isoproterenol in 14 patients with symptomatic chronic obstructive lung disease (COLD) was evaluated in a double-blind crossover 16-week study. FEV1, FVC and SGaw were measured before and 45 minutes after bronchodilator therapy every two weeks, while arterial blood gases were measured every eight weeks, before and 45 minutes after bronchodilator therapy. When the patients were considered as a group, there was no significant difference in mean symptom scores or objective pulmonary functions during the drug and placebo periods. Four patients had significantly higher (p less than .05) and two patients significantly lower mean values for at least one of the pulmonary function tests during the isoproterenol period. The patient who is most likely to benefit from isoproterenol on a regular basis appears to have the following characteristics; (1) consistent improvement in pulmonary function tests 45 minutes after use of nebulized bronchodilator; (2) moderate rather than severe COLD; and (3) a relatively normal DLCO.

Effect of air-supported, continuous, postural oscillation on the risk of early ICU pneumonia in nontraumatic critical illness.

STUDY OBJECTIVES: We hypothesized that continuous, automatic turning utilizing a patient-friendly, low air loss surface would reduce the incidence of early ICU pneumonia in selected groups of critically ill medical patients. DESIGN: Prospective, randomized, controlled clinical trial. SETTING: Medical ICU of a large community teaching hospital. PATIENTS: One hundred twenty-four critically ill new admissions to the medical ICU at Charity Hospital in New Orleans. INTERVENTIONS: Patients were prospectively randomized within one of five diagnosis-related groups (DRG)--sepsis (SEPSIS), obstructive airways disease (OAD), metabolic coma, drug overdose, and stroke--to either routine turning on a standard ICU bed or to continuous turning on an oscillating air-flotation bed for a total of five days. MEASUREMENTS AND RESULTS: Patients were monitored daily during the treatment period for the development of pneumonia. The incidence of pneumonia during the first five ICU days was 22 percent in patients randomized to the standard ICU bed vs 9 percent for the oscillating bed (p = 0.05). This treatment effect was greatest in the SEPSIS DRG (23 percent vs 3 percent, p = 0.04). Continuous automatic oscillation did not significantly change the number of days of required mechanical ventilation, ICU stay, hospital stay, or hospital mortality overall or within any of the DRGs. CONCLUSIONS: We conclude that air-supported automated turning during the first five ICU days reduces the incidence of early ICU pneumonia in selected DRGs; however, this form of automated turning does not reduce other measured clinical outcome parameters.

Effect of prostacyclin on microvascular pressures in a patient with pulmonary veno-occlusive disease.

Continuous-infusion prostacyclin improves symptom scores and decreases mortality in patients with primary pulmonary hypertension, but use of prostacyclin in patients with pulmonary veno-occlusive disease may precipitate pulmonary edema. A patient with pulmonary veno-occlusive disease received a graduated intravenous infusion of prostacyclin and pulmonary capillary pressures were calculated during prostacyclin dose ranging. Calculated capillary pressure increased with low-dose prostacyclin (< or = 6 ng/kg/min) but decreased with higher doses. These data suggest that the post-capillary pulmonary venules in our patient had reversible vasomotor tone, but required a higher dose of prostacyclin to vasodilate than did the precapillary arterioles.

Transbronchial needle aspiration for diagnosis of lung cancer.

Thirty-two consecutive patients with mediastinal lesions suggestive of bronchogenic carcinoma underwent transbronchial needle aspiration. Eighteen of 20 patients (90 percent) with proved bronchogenic carcinoma had malignant cytology specimens or tissue fragments. Of 12 patients with normal cytology specimens, six were subsequently proved to have nonneoplastic disease. Transbronchial needle aspiration appears to offer a sensitive and specific alternative to more invasive surgical techniques used in the diagnosis of malignancies with mediastinal involvement.

Analysis of responses to endothelins in the rabbit pulmonary and systemic vascular beds.

The effects of two isoforms of human endothelin (ET) on the pulmonary and systemic vascular beds were compared in the anesthetized intact-chest rabbit under conditions of constant pulmonary blood flow and left atrial pressure. Intralobar bolus injections of ET-1 (0.1-1 micrograms) and ET-3 (1-3 micrograms) produced modest vasoconstriction in the pulmonary vascular bed, whereas both peptides decreased systemic arterial pressure. The pulmonary vasoconstrictor response to ET-1 and ET-3 was inhibited by intralobar infusion of nitrendipine but was not altered by indomethacin. In contrast to the small effects of ET-1 and ET-3 on intact pulmonary resistance vessels, both peptides markedly contracted isolated pulmonary conductance vessels, with greater activity on venous than on arterial segments. Intravenous bolus injection of ET-1 (0.1-0.3 micrograms) or ET-3 (0.3-1 microgram) decreased systemic arterial pressure, increased cardiac output, and markedly decreased systemic vascular resistance. Higher doses of ET-1 produce a biphasic systemic vascular response with a prominent secondary pressor component. The present data suggest that the pulmonary vasoconstrictor activity of ET-1 is greater than that of ET-3 and their pressor activity depends on an extracellular source of calcium. The pulmonary and systemic hemodynamic effects of ET-1 and ET-3 in the rabbit do not depend on cyclooxygenase products. The systemic vasodilator response to ET-1 is not altered by first-pass lung transit. Furthermore the systemic vasodilator response to both peptides occurs independent of activation of muscarinic, beta 2-adrenergic, and platelet-activating factor receptors. Although ET-1 and ET-3 were initially reported as vasoconstrictor peptides, the present data suggest that, by having unique and potent systemic vasodilator activity, ET-1 and ET-3 act differently in the systemic and pulmonary vascular beds under resting conditions in the rabbit.

Effect of dichloroacetate on mechanical performance and metabolism of compromised diaphragm muscle.

We investigated the effect of dichloroacetate (DCA) on tension generation and carbohydrate metabolism of the rat diaphragm in vitro. Isolated diaphragms were placed in individual organ chambers and were hooked to force-displacement transducers. Net lactate production and glucose and lactate oxidation were measured in vitro. Diaphragmatic fatigue was precipitated by in vivo endotoxemic shock, by in vitro hypoxia, or by in vitro repetitive tetanic stimulation. In diaphragms isolated from endotoxemic rats, DCA increased tension generation by 30 and 20% at stimulation frequencies of 20 and 100 Hz, respectively. Associated with changes in mechanical performance, DCA reduced net lactate production by 53% after 60 min of incubation and increased glucose oxidation 54% but had no effect on lactate oxidation. During in vitro hypoxia, DCA reduced net diaphragmatic lactate production by 30% and increased glucose oxidation by 45% but did not attenuate hypoxic fatigue. DCA had no effect on tension generation during repetitive tetanic stimulation. We conclude that DCA improves in vitro diaphragmatic fatigue due to endotoxicosis but not due to hypoxia or repetitive stimulation.

Endothelin produces pulmonary vasoconstriction and systemic vasodilation.

Endothelin is a newly described polypeptide derived from endothelial cells. The effects of porcine endothelin on the pulmonary vascular bed and systemic vascular bed were investigated in the anesthetized, intact-chest cat under conditions of constant pulmonary blood flow and left atrial pressure. Intralobar bolus injections of porcine endothelin (100-1000 ng) produced a mild vasoconstrictor response in the pulmonary vascular bed. The pulmonary vasoconstrictor response to endothelin was not altered when pulmonary vasomotor tone was increased by infusion of U46619. In contrast to this mild pulmonary vasoconstrictor response, endothelin decreased systemic arterial pressure. Moreover, injections of porcine endothelin into the right and left atria produced similar reductions in aortic pressure as well as similar increases in cardiac output and decreases in systemic vascular resistance. The systemic vasodilator response to porcine endothelin was not affected by beta 2-adrenoceptor blockade. The present data suggest that endothelin does not undergo significant first-pass pulmonary metabolism. The pulmonary vasoconstrictor response to bolus injections of porcine endothelin is not altered by changes in pulmonary vasomotor tone. In contrast, endothelin markedly dilated the systemic vascular bed independently of activation of beta 2-adrenoceptors. The present study provides the first report of the activity of endothelin on pulmonary and systemic hemodynamics in vivo. Moreover, the potent vasodilator activity of endothelin in the systemic vascular bed and its weak effect on pulmonary vessels suggest that endothelin may be more important in the regulation of peripheral vasomotor tone than the pulmonary vascular bed.

Thromboxane-induced pulmonary vasoconstriction: involvement of calcium.

Infusion of tert-butyl hydroperoxide (t-bu-OOH) or arachidonic acid into rabbit pulmonary arteries stimulated thromboxane B2 (TxB2) production and caused pulmonary vasoconstriction. Both phenomena were blocked by cyclooxygenase inhibitors or a thromboxane synthase inhibitor. The increase in pulmonary arterial pressure caused by either t-bu-OOH or arachidonic acid infusion correlated with the concentration of TxB2 in the effluent perfusate. The concentration of TxB2 in the effluent perfusate, however, was always 10-fold greater after arachidonic acid infusion. In the rabbit pulmonary vascular bed lipoxygenase products did not appear involved in the vasoactive response to t-bu-OOH or exogenous arachidonic acid infusion. Calcium entry blockers or a calcium-free perfusate prevented the thromboxane-induced pulmonary vasoconstriction. Calmodulin inhibitors also blocked the pulmonary vasoconstriction induced by t-bu-OOH without affecting the production of TxB2 or prostacyclin. These results suggest that thromboxane causes pulmonary vasoconstriction by increasing cytosol calcium concentration.

Tumor necrosis factor-alpha inhibits endothelium-dependent relaxation.

Tumor necrosis factor-alpha (TNF-alpha) stimulates nitric oxide (NO) in vascular endothelium by induction of the enzyme NO synthase II (NOS II). We examined the effects of TNF-alpha on 1) endothelium-dependent (EDR) and endothelium-independent (EIR) relaxation and 2) contraction of bovine intralobar pulmonary arteries (BPA) and veins (BPV) in vitro. Acetylcholine (ACh), bradykinin (BK), histamine, and A23187 produced EDR of BPA contracted with a 50% effective concentration of U-46619 (15 nM), because relaxation was abolished by endothelium-rubbing and attenuated by L-NG-mono-methylarginine (L-NMMA; 300 microM). TNF-alpha (0.00417, 0.0417, 0.417, and 1.25 micrograms/ml) incubated with BPA for 60 min inhibited EDR of the BPA to ACh, BK, and histamine. The effects of TNF required 30 min for onset. Recovery of EDR occurred 3-4 h after washout of TNF-alpha. Pentoxifylline (1 microM) did not affect ACh-induced EDR but selectively reversed TNF-alpha-mediated inhibition of ACh-induced EDR. TNF-alpha-mediated inhibition of EDR was not reversible by L-NMMA, an inhibitor of NOS I and NOS II, the cyclooxygenase inhibitor ibuprofen, or CV-3908 (1 microM), a platelet-activating factor antagonist. The inhibitory effect of TNF-alpha on EDR was not mediated by nonspecific sensitization of the endothelium to human protein because recombinant human granulocyte colony-stimulating factor (10, 50, and 500 x 10(3) U/ml) did not affect EDR of BPA. The effect of TNF-alpha was specific for release of NO from the endothelium of BPA because TNF-alpha did not affect 1) EDR of BPV to ACh, BK, or ATP; 2) EIR of BPA or BPV to nitroprusside; and 3) contraction of either BPA or BPV to KCl, U-46619, histamine, norepinephrine, or serotonin. Thus TNF-alpha appears to selectively inhibit receptor-mediated EDR and NO release in BPA. TNF-alpha-mediated inhibition of EDR differs from that of L-arginine-based inhibitors and may represent an endogenous physiological mechanism of regulation of NO in the endothelium.