Clinically relevant concentrations of lidocaine and ropivacaine inhibit TNFα-induced invasion of lung adenocarcinoma cells in vitro by blocking the activation of Akt and focal adhesion kinase.

BACKGROUND: Matrix-metalloproteinases (MMP) and cancer cell invasion are crucial for solid tumour metastasis. Important signalling events triggered by inflammatory cytokines, such as tumour necrosis factor α (TNFα), include Src-kinase-dependent activation of Akt and focal adhesion kinase (FAK) and phosphorylation of caveolin-1. Based on previous studies where we demonstrated amide-type local anaesthetics block TNFα-induced Src activation in malignant cells, we hypothesized that local anaesthetics might also inhibit the activation and/or phosphorylation of Akt, FAK and caveolin-1, thus attenuating MMP release and invasion of malignant cells. METHODS: NCI-H838 lung adenocarcinoma cells were incubated with ropivacaine or lidocaine (1 nM-100 µM) in absence/presence of TNFα (20 ng ml(-1)) for 20 min or 4 h, respectively. Activation/phosphorylation of Akt, FAK and caveolin-1 were evaluated by Western blot, and MMP-9 secretion was determined by enzyme-linked immunosorbent assay. Tumour cell migration (electrical wound-healing assay) and invasion were also assessed. RESULTS: Ropivacaine (1 nM-100 µM) and lidocaine (1-100 µM) significantly reduced TNFα-induced activation/phosphorylation of Akt, FAK and caveolin-1 in NCI-H838 cells. MMP-9 secretion triggered by TNFα was significantly attenuated by both lidocaine and ropivacaine (half-maximal inhibitory concentration [IC50]=3.29×10(-6) M for lidocaine; IC50=1.52×10(-10) M for ropivacaine). The TNFα-induced increase in invasion was completely blocked by both lidocaine (10 µM) and ropivacaine (1 µM). CONCLUSIONS: At clinically relevant concentrations both ropivacaine and lidocaine blocked tumour cell invasion and MMP-9 secretion by attenuating Src-dependent inflammatory signalling events. Although determined entirely in vitro, these findings provide significant insight into the potential mechanism by which local anaesthetics might diminish metastasis.

Regional anaesthesia and cancer metastases: the implication of local anaesthetics.

Clinical and basic science studies have demonstrated the anti-inflammatory properties of local anaesthetics. Recent studies have begun to unravel molecular pathways linking inflammation and cancer. Regional anaesthesia is associated in some retrospective clinical studies with reduced risk of metastasis and increased long-term survival. The potential beneficial effects of regional anaesthesia have been attributed mainly to the inhibition of the neuroendocrine stress response to surgery and to the reduction in the requirements of volatile anaesthetics and opioids. Because cancer is linked to inflammation and local anaesthetics have anti-inflammatory effects, these agents may participate in reducing the risk of metastasis, but their mechanism of action is unknown. We demonstrated in vitro that amide local anaesthetics attenuate tumour cell migration as well as signalling pathways enhancing tumour growth and metastasis. This has provided the first evidence of a molecular mechanism by which regional anaesthesia might inhibit or reduce cancer metastases.

Fine epitope mapping of monoclonal antibodies 9B9 and 3G8 to the N domain of angiotensin-converting enzyme (CD143) defines a region involved in regulating angiotensin-converting enzyme dimerization and shedding.

A panel of monoclonal antibodies (mAbs) raised against both the N and C domains of angiotensin-I-converting enzyme (ACE, peptidyl dipeptidase, EC 3.4.15.2) have been extensively mapped and have facilitated the study of various aspects of ACE structure and biology. In this study, we characterize two mAbs, 9B9 and 3G8, that recognize the N domain of ACE and that influence shedding and dimerization. Fine epitope mapping was performed, which mapped the epitopes for these mAbs to the N terminal region of the N domain where they overlap to a large extent, despite having different effects on ACE processing. The mAb 3G8 epitope appears to be shielded by the C domain and to be carbohydrate dependent as binding increased significantly as a result of underglycosylation, whereas these factors did not influence mAb 9B9 recognition. Three mutations within the overlapping region of these two epitopes, Q18H, L19E, and Q22A, which decreased mAb 3G8 binding to the soluble N domain, were introduced into full-length somatic ACE (sACE) to determine their influence on ACE expression and processing. Increased ACE expression, cell surface expression, and basal shedding were observed with all three mutations. Furthermore, cross-linking and western blotting of Chinese hamster ovary (CHO) cell lysates detected two distinct ACE dimers, a native and cross-linked dimer. Increasing amounts of the cross-linked dimer were observed for the mutant sACEQ22A, further implicating the overlapping region of the mAb 9B9 and 3G8 epitopes in ACE processing.

Epitope mapping of mAbs to denatured human testicular ACE (CD143).

Angiotensin I-converting enzyme (ACE; CD143) has two homologous enzymatically active domains (N and C) and plays a crucial role in blood pressure regulation and vascular remodeling. A wide spectrum of monoclonal antibodies (mAbs) to different epitopes on the N and C domains of human ACE have been used to study different aspects of ACE biology. In this study, we characterized a set of nine mAbs, developed against the C domain of human ACE, which recognize the denatured forms of ACE and thus are suitable for the detection and quantification of somatic ACE (sACE) and testicular ACE (tACE) using Western blotting and immunohistochemistry on paraffin-embedded human tissues. The epitopes for these mAbs were defined using species cross-reactivity, phage display library screening, Western blotting and ACE mutagenesis. Most of the mAbs recognized common/overlapping region(s) on both somatic and testicular forms of human ACE, whereas mAb 4E10 was relatively specific for the testicular isoform and mAb 5B9 mainly recognized the glycan attached to Asn 731. This set of mAbs is useful for identifying even subtle changes in human ACE conformation because of denaturation. These mAbs are also sensitive tools for the detection of human sACE and tACE in biological fluids and tissues using proteomic approaches. Their high reactivity in paraffin-embedded tissues provides opportunities to study changes in the pattern of ACE expression and glycosylation (particularly with mAb 5B9) in different tissues and cells.

Connective tissue degradation by invasive rat bladder carcinomas: action of nonspecific proteinases on collagenous matrices.

The invasive RBTCC-8 rat bladder carcinoma cell line (passage number, greater than 100) and its derivates, the RBTCC-8 tumor isografts and the 1-RBTCC-8 daughter cell line (fourth passage), express proteolytic activities of broad substrate specificity, which allow them to efficiently degrade extracellular (collagenous) matrices. Cell-associated, collagenolytic activity is evidenced by the release of hydroxyproline from collagen substrates of types I and IV, by visualizing the low-molecular-weight collagen breakdown products on sodium dodecyl sulfate-polyacrylamide gels, and by the depth of invasion into extracellular matrices in our bone invasion assays. Fractionated by diethylaminoethyl column chromatography, the major collagenolytic activities against collagens of types I and IV coelute in a relatively narrow peak within a NaCl gradient. The pooled collagenolytic diethylaminoethyl fractions contain: (a) two chymotrypsin-like, catheptic activities; (b) activity against a synthetic elastase substrate; (c) gelatinase activity; and (d) caseinolytic activity. Despite efficient collagenolysis, a vertebrate-type collagenase cannot be detected in any of our tumor samples, even after trypsin activation of the tumor cell extracts. The mechanism of action of these nonspecific proteinases is thought to be that of collagen "crosslinkases." The neutral proteinase activities are highest in RBTCC-8 tumor isografts, intermediate in the fourth passage 1-RBTCC-8 carcinoma cell line, and lowest in the RBTCC-8 carcinoma cell line of high passage number. The levels of these nonspecific enzyme activities are well correlated with the depth of invasion into bony matrices, as shown by our invasion assays.

Acute hemodynamic effects of norepinephrine inhibition in patients with severe chronic congestive heart failure.

The pathophysiologic role of high levels of circulating catecholamines in patients with congestive heart failure remains unclear. To assess the hemodynamic contribution of circulating catecholamines, metyrosine (alpha-methyl-p-tyrosine), an inhibitor of catecholamine synthesis, was administered to nine patients with acutely decompensated chronic congestive heart failure. Baseline left ventricular ejection fraction averaged 23.3 +/- 9.9%, whereas cardiac output averaged 3.69 +/- 1.03 liters/min, with a pulmonary wedge pressure of 27.4 +/- 8.5 mm Hg. After 48 h of metyrosine administration, plasma norepinephrine concentration decreased from 919.4 +/- 810.6 to 335.4 +/- 143.1 pg/ml (p less than 0.05). Plasma epinephrine concentration averaged 176.4 +/- 166.0 pg/ml at baseline, and was unchanged during metyrosine administration. Despite the significant decrease in circulating norepinephrine, no significant hemodynamic changes were observed during metyrosine administration. These results suggest that high levels of circulating norepinephrine may be more a marker of severe congestive heart failure than an important contributor to the underlying pathophysiology at this advanced stage of the disease process.

Elastase and neutral cathepsin production by human fibroblasts: effect of culture conditions on synthesis and secretion.

Fibroblasts from normal adult forearm skin and neonatal foreskin were cultured and examined for their ability to synthesize and secrete elastase and neutral cathepsin. All of the cultures examined produced detectable amounts of elastase using insoluble elastin as substrate. An enzyme was also found that hydrolyzed the synthetic elastin substrate, N-succinyl-(Ala)3-p-nitroanilide, but did not degrade insoluble elastin. In addition, activity against the synthetic cathepsin substrate N-benzoyl-DL-phenylalanine-naphthyl ester was found. Inhibitor profiles indicate that the elastin and N-succinyl-(Ala)3-p-nitroanilide degrading activities are due to metalloproteinases. Degradation of N-benzoyl-DL-phenylalanine-naphthyl ester can be inhibited by phenylmethylsulfonyl fluoride. These proteinases were usually found associated with the cell layer. Although activities of the measured proteinases were detected in all cultures, increased or decreased enzyme activities were not predictably related to passage number or length of serum starvation. Degree of confluence also affected proteinase activities. Separation of the dermal-epidermal junction can be produced by the injection of these proteinases into intact mouse skin.

Relation between hemodynamic and ventilatory responses in determining exercise capacity in severe congestive heart failure.

The cause of exercise intolerance in congestive heart failure is unclear. Hemodynamic and ventilatory responses were measured during symptomatic maximal upright bicycle exercise in 28 patients with chronic severe left ventricular failure who achieved a maximal oxygen uptake of only 12 +/- 4 ml/min/kg (+/- standard deviation). All patients reached anaerobic metabolism as the respiratory exchange ratio rose and arterial pH fell significantly. Pulmonary capillary wedge pressure increased from 20 +/- 10 mm Hg at rest to 38 +/- 9 mm Hg at peak exercise and cardiac index increased from 2.51 +/- 0.73 to 4.54 +/- 1.65 liters/min/m2 (both p less than 0.001). Systemic vascular resistance decreased, but pulmonary vascular resistance did not change during exercise. Despite the marked pulmonary venous hypertension at peak exercise, blood gases were unchanged (PaO2, 96 +/- 15 mm Hg; PaCO2, 35 +/- 7 mm Hg). Systemic arterial oxygen content increased from 16 +/- 2 to 17 +/- 2 vol% (p less than 0.01). Changes in pulmonary capillary wedge pressure did not correlate with changes in arterial oxygen content. Results were similar whether patients were limited by dyspnea or fatigue. Thus, exercise intolerance in patients with severe left ventricular failure is associated with marked elevation of pulmonary capillary wedge pressure and anaerobic metabolism without hypoxemia or altered carbon dioxide tension. These findings suggest that exercise ability in congestive heart failure is more dependent on cardiac output than on ventilatory consequences of pulmonary congestion.

Propofol-induced pancreatitis: recurrence of pancreatitis after rechallenge.

We report a case of pancreatitis, which occurred while the patient was on a propofol drip and then recurred after resolution following an inadvertent rechallenge with propofol. The initial episode was associated with hypertriglyceridemia, whereas the latter was not. The association between propofol and pancreatitis is definite and may occur independently of significant hypertriglyceridemia.

Assessment of routine chest roentgenograms and the physical examination to confirm endotracheal tube position.

We consecutively and prospectively studied 219 critically ill patients to evaluate the accuracy of the physical examination in assessing ETT position and the appropriateness of taking routine chest x-ray films after intubation in the ICU. As a result of x-ray findings, 14 percent of the patients required ETT repositioning, and 5 percent had main-stem intubations. Endobronchial intubation was more common in females than in males, and frequently occurred after emergency intubations. Sixty percent of the main-stem intubations occurred despite the presence of equal breath sounds on examination. Techniques to minimize the risk of tube malposition, such as cuff ballottement in the suprasternal notch and referencing the ETT centimeter markings, were not completely reliable. This study confirms the unreliability of the physical examination to assess ETT position. Chest x-ray films after intubation are indicated to verify tube position, particularly after emergency intubations. Other techniques such as use of a lighted stylet require evaluation to determine whether they are more cost-effective in verifying ETT placement in patients who have no other indication for postintubation x-ray films.

Molecular characteristics of the inhibition of human neutrophil elastase by nonsteroidal antiinflammatory drugs.

Nonsteroidal antiinflammatory drugs(NSAIDs) are known as clinically effective agents for treatment of inflammatory diseases. Inhibition of cyclooxygenase has been thought to be a major facet of the pharmacological mechanism of NSAIDs. However, it is difficult to ascribe the antiinflammatory effects of NSAIDs solely to the inhibition of prostaglandin synthesis. Human neutrophil elastase (HNElastase; HNE, EC 3.4.21.37) has been known as a causative factor in inflammatory diseases. To investigate the specific relationship between HNElastase inhibition and specificity of molecular structure of several NSAIDs, HNElastase was purified by Ultrogel AcA54 gel filtration, CM-Sephadex ion exchange, and HPLC (with TSK 250 column) chromatography. HNElastase was inhibited by aspirin and salicylate in a competitive manner and by naproxen, ketoprofen, phenylbutazone, and oxyphenbutazone in a partial competative manner, but not by ibuprofen and tolmetin. HNElastase-phenylbutazone-complex showed strong Raman shifts at 200, 440, 1124, 1194, 1384, 1506, and 1768 cm(-1). The Raman bands 1194, 1384, and 1768 cm(-1) may represent evidences of the conformational change at -N=N-phi radical, pyrazol ring, and -C=O radical of the elastase-drug complex, respectively. Phenylbutazone might be bound to HNElastase by ionic and hydrophobic interaction, and masked the active site. Inhibition of HNElastase could be another mechanism of action of NSAIDs besides cyclooxygenase inhibition in the treatment of inflammatory diseases. Different inhibition characteristics of HNE-lastase by NSAIDs such as aspirin, phenylbutazone-like drugs and ineffective drugs could be important points for drawing the criteria for appropriate drugs in clinical application.

Ionizing radiation induces early, sustained increases in collagen biosynthesis: a 48-week study in mouse skin and skin fibroblast cultures.

Groups of 10 CF1 female mice, irradiated to the thorax with a dual-head 137Cs gamma-RAY source, received single doses of 0, 5, 10, 15, or 25 Gy. One to forty-eight weeks later collagen synthesis was measured in minced skin specimens incubated in medium containing [3H]proline and then assayed for radioactive hydroxyproline. A progressive, generally dose-dependent increase in collagen biosynthesis, up to 50% above control sites, was found 1, 4, and 12 weeks after radiation exposure. These changes showed further small fluctuations at 12-36 weeks, increasing again at the 48-week interval. At the same times throughout the study fibroblasts were cultured from skin explants. Following the second subculture, these cells were also incubated in medium containing [3H]proline, and collagen synthesis was again determined by [3H]hydroxyproline assay. At all radiation dose levels studied, collagen production increased threefold by 12 weeks postradiation and remained elevated for the 48-week duration of the study. In vitro radiation dose response differences were not observed.

Physical properties of aerosols produced by dermabrasion.

Medical personnel who perform dermabrasions are exposed to airborne blood and tissue fragments. The safety or hazards of exposure to such aerosols have not been adequately studied. Using scanning electron microscopy, the air density and size distribution of particles produced during dermabrasion were analyzed. Such particles are of sufficient size to allow for access to and retention by mucosal and pulmonary surfaces. Transmission electron microscopy reveals amorphous particles without discernible cell membranes. Commonly used personnel protection standards do not prevent respiration of these particulates. Mathematical estimation of particle size production allows extrapolation of these data to other rotary instrument applications.

Management of the difficult airway.

For clinicians involved in airway management, a plan of action for dealing with the difficult airway or a failed intubation should be developed well in advance of encountering a patient in whom intubation is not routine. When difficulty is anticipated, the equipment necessary for performing a difficult intubation should be immediately available. It also is prudent to have a surgeon skilled in performing a tracheotomy and a criothyroidotomy stand by. The intubation should be attempted in the awake state, preferably using the fiberoptic bronchoscope. The more challenging situation is when the difficult airway is confronted unexpectedly. After the first failed attempt at laryngoscopy, head position should be checked and the patient ventilated with oxygen by mask. A smaller styletted tube and possibly a different laryngoscope blade should be selected for a second attempt at intubation. The fiberoptic bronchoscope and other equipment for difficult intubation should be obtained. A second attempt should then be made. If this is unsuccessful, the patient should be reoxygenated, and assistance including a skilled anesthesiologist and surgeon should be summoned. On a third attempt, traction to the tongue can be applied by an assistant, a tube changer could be used to enter the larynx, or one of the other special techniques previously described can be used. If this third attempt fails, it may be helpful to have a physician more experienced in airway management attempt intubation after oxygen has been administered to the patient. If all attempts are unsuccessful, then invasive techniques to secure the airway will have to be performed.

Prospective randomized study of the role of N-acetyl cysteine in reversing doxorubicin-induced cardiomyopathy.

We conducted a randomized prospective trial in 19 disease-free soft tissue sarcoma patients with doxorubicin-induced cardiomyopathy identified by ECG radionuclide angiography at rest and during exercise to determine the efficacy of the free radical scavenger, N-Acetyl Cysteine (NAC), in reversing the drug's cardiotoxic effect. Of the 19 patients, 11 received oral NAC (5.5 gm/m2 daily for 30 days) and eight patients served as controls. Patients were stratified for age less than greater than 45 years, time from final dose of doxorubicin to randomization less than greater than 8 months, and history of treatment with mediastinal irradiation. The two groups were well-matched for all parameters. Cumulative mean doxorubicin dose (523 mg/m2 and 532 mg/m2) and range 500-600 mg/m2 was comparable. Left ventricular (LV) ejection fraction before randomization was not significantly different between the two groups either at rest (39 +/- 10% control, 38 +/- 13% NAC) or during exercise (38 +/- 12% control, 35 +/- 11% NAC). Neither rest nor exercise ejection fraction values changed significantly in either group between prerandomization and 1-month postrandomization studies. Late studies performed in seven NAC patients 3-5 months after randomization revealed no difference in LV ejection fraction compared to 1-month postrandomization values. Clinical course in patients with overt congestive heart failure was similar in both groups. LV function did not return to normal in any patient in either group. We conclude that N-Acetyl Cysteine has no effect in reversing long standing doxorubicin-induced cardiomyopathy.

Bio-markers and the search for extinct life on Mars.

Geologic and climatologic studies suggest that conditions on early Mars were similar to early Earth. Because life on Earth is believed to have originated during this early period (3.5 billion years ago), the Martian environment could have also been conducive to the origin of life. To investigate this possibility we must first define the attributes of an early Martian biota. Then, specific geographic locations on Mars must be chosen where life may have occurred (i.e. areas which had long standing water), and within these distinct locations search for key signatures or bio-markers of a possible extinct Martian biota. Some of the key signatures or bio-markers indicative of past biological activity on Earth may be applicable to Mars including: reduced carbon and nitrogen compounds, CO3(2-), SO4(2-), NO3-, NO2- [correction of NO2(2)], Mg, Mn, Fe, and certain other metals, and the isotopic ratios of C, N and S. However, we must also be able to distinguish abiotic from biologic origins for these bio-markers. For example, abiotically fixed N2 would form deposits of NO3- and NO2-, whereas biological processes would have reduced these to ammonium containing compounds, N2O, or N2, which would then be released to the atmosphere. A fully equipped Mars Rover might be able to perform analyses to measure most of these biomarkers while on the Martian surface.

The use of mineral crystals as bio-markers in the search for life on Mars.

Photographs that depict presumed fluvial features on the martian surface have led geologists to hypothesize that water flowed across the early martian terrain. From this, it has been further hypothesized that the surface and atmospheric conditions on early Mars were similar to those on early Earth. Because the oldest fossil evidence of life on Earth dates back to this early period, at least 3.5 billion years ago, the possibility exists that the early Martian environment could have also been conducive to the origin of life. To investigate this possibility, universal signatures or bio-markers indicative of past (or present) biological activity must be identified for use in the search for life on Mars. Several potentially applicable biomarkers have been identified and include: organics (e.g., specific classes of lipids and hopanes), suites of specific inorganic and organic compounds, as well as the isotopic ratios of C, N, and S. Unfortunately, all of these bio-markers may be of biologic or abiotic origin; these origins are often difficult to distinguish. Thus, the discovery of any one of these compounds alone is not a bio-marker. Because minerals produced under biologic control have distinctive crystallographies, morphologies, and isotopic ratios that distinguishable from abiotically produced minerals with the same chemical composition, and are stable through geologic time, we propose the use of minerals resulting from biologically controlled mineralization processes as bio-markers.

Search for life on Mars: evaluation of techniques.

An important question for exobiology is, did life evolve on Mars? To answer this question, experiments must be conducted on the martian surface. Given current mission constraints on mass, power, and volume, these experiments can only be performed using proposed analytical techniques such as: electron microscopy, X-ray fluorescence, X-ray diffraction, alpha-proton backscatter, gamma-ray spectrometry, differential thermal analysis, differential scanning calorimetry, pyrolysis gas chromatography, mass spectrometry, and specific element detectors. Using prepared test samples consisting of 1% organic matter (bovine serum albumin) in palagonite and a mixture of palagonite, clays, iron oxides, and evaporites, it was determined that a combination of X-ray diffraction and differential thermal analysis coupled with gas chromatography provides the best insight into the chemistry, mineralogy, and geological history of the samples.