Electrical stimulation of the midbrain mediates metastatic tumor growth.

Pulmonary metastases were counted 10 days after female rats received tail-vein injections of Walker-256 carcinosarcoma cells. Previous observations that halothane anesthesia plus hind-limb amputation increases the number of metastases were confirmed. Amputation under the analgesia of electrical stimulation of the midbrain was found to increase metastatic activity. However, the stimulus-produced analgesia alone also increased the number of metastases. Systemically administered naloxone blocked the analgesic effect of midbrain stimulation but did not block the increase in the number of pulmonary metastases.

Neutrophil-induced injury of rat pulmonary alveolar epithelial cells.

The damage to pulmonary alveolar epithelial cells that occurs in many inflammatory conditions is thought to be caused in part by phagocytic neutrophils. To investigate this process, we exposed monolayers of purified rat alveolar epithelial cells to stimulated human neutrophils and measured cytotoxicity using a 51Cr-release assay. We found that stimulated neutrophils killed epithelial cells by a process that did not require neutrophil-generated reactive oxygen metabolites. Pretreatment of neutrophils with an antibody (anti-Mo1) that reduced neutrophil adherence to epithelial cells limited killing. Although a variety of serine protease inhibitors partially inhibited cytotoxicity, we found that neutrophil cytoplasts, neutrophil lysates, neutrophil-conditioned medium, purified azurophilic or specific granule contents, and purified human neutrophil elastase did not duplicate the injury. We conclude that stimulated neutrophils can kill alveolar epithelial cells in an oxygen metabolite-independent manner. Tight adherence of stimulated neutrophils to epithelial cell monolayers appears to promote epithelial cell killing.

Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene.

Impaired fibrinolytic activity within the lung is a common manifestation of acute and chronic inflammatory lung diseases. Because the fibrinolytic system is active during repair processes that restore injured tissues to normal, reduced fibrinolytic activity may contribute to the subsequent development of pulmonary fibrosis. To examine the relationship between the fibrinolytic system and pulmonary fibrosis, lung inflammation was induced by bleomycin in transgenic mice that either overexpressed or were completely deficient in murine plasminogen activator inhibitor-1 (PAI-1). 2 wk after 0.075 U of bleomycin, the lungs of transgenic mice overexpressing PAI-1 contained significantly more hydroxyproline (118 +/- 8 micrograms) than littermate controls (70.5 +/- 8 micrograms, P < 0.005). 3 wk after administration of a higher dose of bleomycin (0.15 U), the lung hydroxyproline content of mice completely deficient in PAI-1 (49 +/- 8 micrograms) was not significantly different (P = 0.63) than that of control animals receiving saline (37 +/- 1 micrograms), while hydroxyproline content was significantly increased in heterozygote (77 +/- 12 micrograms, P = 0.06) and wild-type (124 +/- 19 micrograms, P < 0.001) littermates. These data demonstrate a direct correlation between the genetically determined level of PAI-1 expression and the extent of collagen accumulation that follows inflammatory lung injury. These results strongly support the hypothesis that alterations in fibrinolytic activity influence the extent of pulmonary fibrosis that occurs after inflammatory injury.

Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice.

Mice deleted for the plasminogen activator inhibitor-1 (PAI-1) gene are relatively protected from developing pulmonary fibrosis induced by bleomycin. We hypothesized that PAI-1 deficiency reduces fibrosis by promoting plasminogen activation and accelerating the clearance of fibrin matrices that accumulate within the damaged lung. In support of this hypothesis, we found that the lungs of PAI-1(-/-) mice accumulated less fibrin after injury than wild-type mice, due in part to enhanced fibrinolytic activity. To further substantiate the importance of fibrin removal as the mechanism by which PAI-1 deficiency limited bleomycin-induced fibrosis, bleomycin was administered to mice deficient in the gene for the Aalpha-chain of fibrinogen (fib). Contrary to our expectation, fib(-/-) mice developed pulmonary fibrosis to a degree similar to fib(+/-) littermate controls, which have a plasma fibrinogen level that is 70% of that of wild-type mice. Although elimination of fibrin from the lung was not in itself protective, the beneficial effect of PAI-1 deficiency was still associated with proteolytic activity of the plasminogen activation system. In particular, inhibition of plasmin activation and/or activity by tranexamic acid reversed both the accelerated fibrin clearance and the protective effect of PAI-1 deficiency. We conclude that protection from fibrosis by PAI-1 deficiency is dependent upon increased proteolytic activity of the plasminogen activation system; however, complete removal of fibrin is not sufficient to protect the lung.

Binding of Griffonia simplicifolia I lectin to rat pulmonary alveolar macrophages and its use in purifying type II alveolar epithelial cells.

We report that the isolectin Griffonia simplicifolia I-B4 isolated from G. simplicifolia seeds binds to rat alveolar macrophages present in frozen sections of lung tissue or bronchoalveolar lavage fluid. G. simplicifolia I-B4 does not bind to alveolar epithelial cells. We established that G. simplicifolia I-B4 binds to the macrophages via interaction with terminal alpha-D-galactopyranosyl residues present on these cells. This was substantiated by demonstrating that binding is inhibited either by the haptenic sugar alpha-D-galactopyranoside or by treating the cells with coffee bean alpha-galactosidase. Because murine laminin is known to contain terminal alpha-D-galactopyranosyl end-groups, and because we found that an anti-laminin antiserum binds to rat alveolar macrophages, we suspect that G. simplicifolia I-B4 may be binding to laminin present on the macrophages. To isolate alveolar type II epithelial cells from rat lungs, we developed a method that utilizes the lectin G. simplicifolia I. When proteinase-derived suspensions of pulmonary cells are incubated with G. simplicifolia I, the macrophages agglutinate and can be removed by filtration through nylon mesh. After incubating the resulting cellular suspension in tissue culture, the adherent cells are 94 +/- 2% (S.D.) type II cells. When compared to cells isolated by repeated differential adherence, the lectin-prepared type II cells have similar morphology and staining characteristics, form domes in monolayers and incorporate similar amounts of palmitate into disaturated phosphatidylcholine. We believe that the procedure outlined in this report provides a simple and effective method to isolate type II alveolar epithelial cells from rat lungs.

Treatment of bleomycin-induced pulmonary fibrosis by transfer of urokinase-type plasminogen activator genes.

During acute and chronic inflammatory lung diseases, the normal fibrinolytic activity in the alveolar space is inhibited by increased levels of plasminogen activator inhibitor 1 (PAI-1). Transgenic mice having increased fibrinolytic activity due to genetic deficiency of PAI-1 develop less fibrosis after bleomycin-induced lung inflammation. These observations led us to hypothesize that pulmonary fibrosis could be limited through enhancement of alveolar fibrinolytic activity by adenovirus-mediated transfer of the urokinase-type plasminogen activator (uPA) gene to the lung. To investigate this hypothesis, 0.075 U of bleomycin was introduced intratracheally into mice. Twenty-one days later, the mice were treated intratracheally with phosphate-buffered saline (PBS), a control adenovirus, or adenoviruses containing murine or human uPA cDNAs. On day 28, the mice were sacrificed, and lung fibrosis was quantitated by measuring hydroxyproline content. As expected, bleomycin caused a doubling in lung hydroxyproline to 345.6+/-28.2 microg/lung (SEM) compared with mice receiving PBS (170.2+/-4.0 microg/lung). Treatment of the bleomycin-injured mice with the control adenovirus on day 21 had no impact on lung fibrosis (338.4+/-17.2 microg/lung). Importantly, the human uPA adenovirus significantly reduced (p<0.05) lung hydroxyproline (281.2+/-22.8 microg/lung), thus attenuating by 38% the bleomycin-induced increase in lung collagen. The improvement in bleomycin-induced lung fibrosis resulting from treatment with the human uPA adenovirus further supports the importance of the fibrinolytic system during inflammatory lung injury and repair.

Upregulation of fibrinolysis by adenovirus-mediated transfer of urokinase-type plasminogen activator genes to lung cells in vitro and in vivo.

Impaired fibrinolytic activity within the lungs is a common manifestation of acute and chronic inflammatory lung diseases. Our previous work using transgenic mice showed that upregulation of fibrinolysis reduced pulmonary fibrosis following bleomycin-induced inflammatory lung injury. As a strategy to accelerate fibrinolysis, we generated recombinant adenoviruses containing human and mouse urokinase-type plasminogen activator (uPA) cDNAs. Both vectors induced the expression of functional uPA in human lung-derived epithelial A549 cells. A single intratracheal instillation of these uPA-containing adenoviruses into mouse lungs resulted in increased plasminogen activator activity in bronchoalveolar lavage fluid for at least 2 weeks. Plasma-derived fibrin-rich matrices overlaid on A549 cells infected with these uPA vectors were lysed efficiently in a dose-dependent fashion. Similarly, fibrin matrices formed within intact lungs that had been infected with these uPA-containing adenoviruses were also lysed more rapidly compared with noninfected and control virus-infected lungs. These results indicate that adenovirus-mediated transduction of uPA successfully upregulates fibrinolysis in vitro and in vivo. These uPA vectors can be readily used for testing the role of the fibrinolytic system in animal models of lung fibrosis, with particular attention to their therapeutic potential.

Adenovirus-mediated transfer of the CFTR gene to lung of nonhuman primates: biological efficacy study.

We have evaluated the biological efficacy of E1-deleted adenoviruses in baboons for lung-directed gene therapy of cystic fibrosis (CF). The experimental design attempted to simulate a phase I clinical trial with animals receiving a single dose of virus to an isolated pulmonary segment. A total of 14 animals divided into four groups, each of which received escalating doses of virus, were used. Individual animals were necropsied 4 and 21 days after gene transfer and tissues were carefully surveyed for gene expression. Expression of the transgene was localized primarily to the area into which it was infused; the efficiency of recombinant gene expression and the abundance of transgene sequences were proportional to dose and both diminished with time. Transgene expression was found predominantly in alveolar cells with patches of expression in the proximal and distal airway. Analysis of adenoviral protein expression within transgene-expressing cells revealed infrequent expression of the E2a gene and no detectable expression of late genes (i.e., fiber protein). These results suggest that recombinant adenovirus can be used to transfer genes efficiently to the lung of nonhuman primates and that therapeutic strategies of cystic fibrosis may require repetitive administration with current vectors.

Adenovirus-mediated transfer of the CFTR gene to lung of nonhuman primates: toxicity study.

In preparation for human trials of gene therapy for cystic fibrosis (CF), we performed a preclinical study of gene transfer into the lungs of baboons. Recombinant adenovirus vectors containing expression cassettes for human cystic fibrosis transmembrane conductance regulator (CFTR) and Escherichia coli beta-galactosidase (lacZ) were instilled through a bronchoscope into limited regions of lung in 14 baboons. A detailed accounting of the extent, distribution, and duration of gene expression is contained in a companion article (Engelhardt et al., 1993b). In this article, we report the results of toxicity studies in which clinical laboratory tests, chest radiographs, and necropsy studies were used to detect adverse effects. The only adverse effect noted was a mononuclear cell inflammatory response within the alveolar compartment of animals receiving doses of virus that were required to induce detectable gene expression. Minimal inflammation was seen at 10(7) and 10(8) pfu/ml, but at 10(9) and more prominently at 10(10) pfu/ml, a perivascular lymphocytic and histiocytic infiltrate was seen. The intensity of inflammation increased between 4 and 21 days. At its greatest intensity, there was diffuse alveolar wall damage with intra-alveolar edema. Airways were relatively spared, despite the intensity of alveolar inflammation. Clinical tests did not accurately reflect the presence of lung inflammation, with the exception of chest radiographs which revealed alveolar infiltrates, but only in regions of lung having the greatest intensity inflammation. We conclude that adenovirus-mediated gene transfer into the lungs of baboons is associated with development of alveolar inflammation at high doses of virus.

Pulmonary inflammation induced by incomplete or inactivated adenoviral particles.

One of the major obstacles to pulmonary-directed gene therapy using adenoviral vectors is the induction of inflammation. We investigated whether the adenoviral particles that constitute the initial inoculum can serve as an inflammatory stimulus, independent of their ability to express genes that they contain. Viral particles were prepared that are defective in gene expression by (i) isolating particles that have incomplete genomes by selecting those that have buoyant densities on CsCl density gradients lighter than complete viruses; and (ii) cross-linking viral DNA by exposure to ultraviolet light in the presence of 8-methoxypsoralen. The defective particles retained their icosahedral appearance when viewed by electron microscopy but lost their plaque-forming ability on 293 cells. High doses of intact, incomplete, or inactivated viral particles were instilled intratracheally into CBA/J mice, and after 6 days the amount of inflammation was quantified by counting inflammatory cells contained within lung tissue. We found that the inflammatory responses induced by the incomplete or inactivated viral vectors were quantitatively similar to those caused by intact, competent viral vectors. We conclude that high doses of adenoviral vectors that are used for gene therapy can induce pulmonary inflammation, independent of expressing the genes they contain.

Adult respiratory distress syndrome in neutropenic patients.

The precise pathophysiologic mechanisms that cause the adult respiratory distress syndrome are unknown. Indirect evidence from human studies and extrapolations from animal models have suggested that phagocytic neutrophils are important in the pathogenesis of this disease. To further evaluate the role of neutrophils, the frequency of neutropenia in 18 bacteremic patients who had the adult respiratory distress syndrome was compared with that in a control group who had bacteremia alone. Three of 18 patients in the group with the adult respiratory distress syndrome were neutropenic as opposed to one of 18 in the control group (p greater than 0.6). Histologic examination of the lungs from two patients with the adult respiratory distress syndrome and neutropenia demonstrated the absence of neutrophils. It is likely that there are many pathways that lead to the adult respiratory distress syndrome. Although neutrophils may be involved in some of these processes, this study demonstrates that neutrophils are not required for the development of the syndrome. In the appropriate clinical setting, the diagnosis of the adult respiratory distress syndrome should not be excluded solely because of neutropenia.

Elevation of serum prostaglandin E levels following electrical stimulation of the midbrain.

Serum prostaglandin E (PGE) levels were measured in rats immediately following electrical stimulation of the mesencephalic periaqueductal gray (PAG) region and the nucleus raphe magnus (NRM) and compared to controls. An additional group received aspirin prior to PAG stimulation. A significant increase in serum PGE levels was found after stimulation of the PAG, but not the NRM. Aspirin inhibited the stimulation-induced increases in PGE.

Pulse oximetry for tapering supplemental oxygen in hospitalized patients. Evaluation of a protocol.

In a randomized study, we determined the clinical and financial effects of replacing arterial blood gas measurements with finger pulse oximeter readings during the process of tapering supplemental oxygen in hospitalized patients. The 16 patients in the control group, whose management followed conventional practice in our hospital, received a total of 57 arterial blood gas measurements during the 6.6 (mean) days it took for them to taper to their discharge supplemental oxygen level (usually room air). The 13 patients randomized to the oximeter study group had their arterial oxygen saturation monitored by pulse oximetry. The physicians of patients in the oximeter group were at liberty to obtain arterial blood gas determinations during the study if they desired. The oximeter study group had fewer (p less than 0.005) arterial punctures for blood gas measurements (total of 16 for the group) and fewer (p less than 0.001) days on supplemental oxygen (mean of 2.7 days per patient). We conclude that substituting noninvasive pulse oximetry for arterial blood gas measurements during reductions of supplemental oxygen shortened the days of oxygen use and decreased the number of arterial blood gas determinations in our patients. In addition to reducing the discomfort to patients, the use of oximetry was of financial benefit in that it reduced medical personnel time, blood gas analyzer use, and duration of oxygen administration.

Evidence for adenosine triphosphate degradation in critically-ill patients.

Alterations of cellular energy metabolism may provide important markers during the clinical course of critically ill patients. To determine whether adenosine triphosphate (ATP) degradation occurs in critically ill patients, we measured levels of adenosine, inosine, hypoxanthine, and xanthine in 18 patients and seven control subjects. The mean concentration of hypoxanthine (3.8 microM) in the critically ill patients was elevated (p less than 0.01) compared to that of our control population (0.1 microM). A subgroup of seven critically ill patients had levels of hypoxanthine, xanthine, or inosine higher than those of any member of the control group. This subgroup was characterized by a lower systolic blood pressure, an increased requirement for vasopressors, and a markedly decreased survival rate when compared to the other critically ill patients. Arterial and mixed venous blood gas values were not helpful in predicting survival and did not correlate with levels of ATP degradation products. In two patients who showed subsequent clinical improvement, the initially elevated levels of hypoxanthine and xanthine returned to normal. This study indicates that critically ill patients have elevated levels of ATP degradation products. These increased levels may indicate cellular hypoxia.

Detection of experimental rat liver tumors by contrast-assisted ultrasonography.

RATIONALE AND OBJECTIVES: The authors characterized the effect of intravenous lipid-coated microbubbles (LCMs) on the echogenicity of malignant liver tumors. METHODS: Novikoff hepatoma cells were inoculated into the livers of 16 anesthetized Sprague-Dawley rats. Sonograms were obtained weekly after tail-vein injection with either 0.2 mL/kg LCMs or saline control. RESULTS: A statistically significant difference in the signal-to-noise ratio (SNR) was observed between the group that received LCMs (10 rats) and the group that received saline (6 rats) (P < .01). The effect persisted for 30 minutes after contrast injection. Selective leakage and accumulation of LCMs into the tumor tissue itself was confirmed histologically using lipid-specific counterstains. CONCLUSIONS: Intravenous injection of the LCM contrast agent produces a rapid increase in the echogenicity of the experimental Novikoff tumor in the rat liver.

Quantitative assessment of tumor enhancement by ultrastable lipid-coated microbubbles as a sonographic contrast agent.

We have previously reported that ultrastable lipid-coated microbubbles make a suitable ultrasonic contrast agent in the brain, causing increased intensity of echoes that persists for many hours. We showed that intravenously administered lipid-coated microbubbles accumulate selectively in rat brain gliomas with echogenicity enhancement for up to 1 hour, allowing visualization of the growing lesions 40% (2 days) earlier than can be seen without contrast. This work is a detailed evaluation of the accumulation of the lipid-coated microbubbles in tumor and the effect of the bubbles on the echogenicity of insonified tumors. Using a lipid-specific stain, we measured and characterized the distribution of the bubbles in the brain and tumor. We showed that on the scan, the enhancement of the tumor is accompanied by a change in the signal-to-noise ratio of the echoes from the tumor. We identified characteristic textural changes associated with contrast-enhanced tumor using spectral analysis.

Lipid-coated ultrastable microbubbles as a contrast agent in neurosonography.

Lipid-coated microbubbles can be synthesized from selected lipid monolayer systems for use as ultrasonic contrast. These microbubbles have the property of longevity of weeks in vitro (ultrastability) and longevity of hours in vivo. The bubbles can be manufactured with a mean diameter of approximately 2 microns in a tight diameter distribution; all are less than 6 microns and 99% are smaller than 4.5 microns. The current study compared the in vivo survival characteristics of these lipid-coated microbubbles with microbubbles produced by saline. The comparison was made in the rat brain using direct intraparenchymal injections and injections into a previously created cyst/coagulum. The echogenic enhancement by the lipid-coated microbubbles persisted in vivo for over 24 hours in both the intraparenchymal environment and in the cyst/coagulum. The saline bubble echos were not detectable by 3 hours in a cyst/coagulum, and not detectable in the parenchyma after 2 hours. The sonographic characteristics and longevity of lipid-coated microbubbles make this agent a potentially useful clinical contrast material for neurosonography.

Effect of midbrain stimulation on amphetamine-induced stereotypy in rats.

Behavioral changes were induced in rats by administration of high doses of amphetamine (5 mg/kg): among these was the development of rapid sniffing. The effect of electrical stimulation of the midbrain periaqueductal gray was then examined. This stimulation is known to release an enkephalin-like substance into the ventricular spinal fluid and to induce analgesia. Stimulation blocked amphetamine-induced sniffing. This effect was blocked in turn by pretreatment with naloxone, a specific opiate antagonist. We discuss this finding in the context of opiate-catecholamine antagonism in the CNS.

Morphine increases metastatic tumor growth.

Walker 256 carcinosarcoma cells produce subpleural pulmonary metastases when given intravenously to the Sprague-Dawley rat. The number of metastases increases when the rat is given morphine subsequent to the tumor load. The increase in the number of metastases can be blocked be pretreatment with the opiate antagonist naloxone. Naloxone itself does not influence the number of metastases. Pentazocine is an opiate that is agonistic to the endorphin kappa-type opiate receptor and partially antagonistic to the mu-type receptor, where morphine acts primarily. While pentazocine alone has no influence on metastases and may decrease the number when given early, pentazocine partially blocks the metastatic inducing effect of morphine.

Beta-endorphin injected into the nucleus of the raphe magnus facilitates metastatic tumor growth.

Electrical stimulation of the periaqueductal gray of the rat's midbrain analgesia leads to an increase in the number of artificial pulmonary metastases from the Walker 256 tumor. In an effort to investigate the influence of the pain suppression system and its associated peptides on this phenomenon, we activated the pain suppression system directly from the Nucleus of the Raphe Magnus, a non-opioid subsystem. After inducing analgesia by direct injection of beta-endorphin on the Nucleus of the Raphe Magnus, we noted an increase in the number of artificial pulmonary metastases. This result could be blocked by pretreatment with naloxone. If the Nucleus of the Raphe Magnus was activated by electrical stimulation sufficient to induce analgesia, the metastatic effect was still present but markedly attenuated.