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.

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.

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.

Gamma Knife treatment of 100 consecutive meningiomas.

Clinical and imaging results of Gamma Knife treatment of 100 consecutive patients with intracranial meningiomas are reported. Only 1 patient refused follow-up imaging and her symptoms remain improved after 1 year. Mean values for the patient and treatment parameters were age 61 years, duration of symptoms 3.6 years, time since diagnosis 3 years, average tumor diameter 2.4 cm, surface radiation dose 15 Gy and number of isocenters 5. Clinical outcomes revealed that 6 were improved, 75 were unchanged and 17 had deteriorated. Of the latter, 8 were operated, 4 were treated medically and 5 died. Imaging follow-up showed no growth in 87 patients. The size of tumors treated ranged from 0.66 to 6.8 cm average diameter. In the 77 patients with tumors with an average diameter of 3 cm or less, only 2 (3%) showed further growth, and none died of tumor-related causes.

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.

Death and other complications of emergency airway management in critically ill adults. A prospective investigation of 297 tracheal intubations.

BACKGROUND: Hospitalized patients outside of the operating room frequently require emergency airway management. This study investigates complications of emergency airway management in critically ill adults, including: (1) the incidence of difficult and failed intubation; (2) the frequency of esophageal intubation; (3) the incidence of pneumothorax and pulmonary aspiration; (4) the hemodynamic consequences of emergent intubation, including death, during and immediately following intubation; and (5) the relationship, if any, between the occurrence of complications and supervision of the intubation by an attending physician. METHODS: Data were collected on consecutive tracheal intubations carried out by the intensive care unit team over a 10-month period. Non-anesthesia residents were supervised by anesthesia residents, critical care attending physicians, or anesthesia attending physicians. RESULTS: Two hundred ninety-seven consecutive intubations were carried out in 238 adult patients. Translaryngeal tracheal intubation was accomplished in all patients. Intubation was difficult in 8% of cases (requiring more than two attempts at laryngoscopy by a physician skilled in airway management). Esophageal intubation occurred in 25 (8%) of the attempts but all were recognized before any adverse sequelae resulted. New infiltrates suggestive of pulmonary aspiration were present on chest radiography after 4% of intubations. Seven patients (3%) died during or within 30 min of the procedure. Five of the seven patients had systemic hypotension (systolic blood pressure < or = 90 mmHg), and four of the five were receiving vasopressors to support systolic blood pressure. Patients with systolic hypotension were more likely to die after intubation than were normotensive patients (P < 0.001). There was no relationship between supervision by an attending physician and the occurrence of complications. CONCLUSIONS: In critically ill patients, emergency tracheal intubation is associated with a significant frequency of major complications. In this study, complications were not increased when intubations were accomplished without the supervision of an attending physician as long as the intubation was carried out or supervised by an individual skilled in airway management. Mortality associated with emergent tracheal intubation is highest in patients who are hemodynamically unstable and receiving vasopressor therapy before intubation.

Synthesis and evaluation of nuclear targeting peptide-antisense oligodeoxynucleotide conjugates.

An endogenous nuclear enzyme, RNase H, is an important component in determining the efficacy of antisense oligodeoxynucleotides (ODNs). In an effort to improve the potency of antisense ODNs, conjugates with three different nuclear targeting signal peptides were prepared. These short peptide sequences have been shown to facilitate transport of macromolecules into the nucleus of cells. Efficient chemistry for the synthesis of ODN-peptide conjugates is described. Reaction of 5'-aminohexyl-modified ODNs with iodoacetic anhydride gave pure iodoacetamide ODNs (IA-ODNs) in good yield. These electrophilic intermediates were reacted with thiol-containing peptides to give ODN-peptides in excellent yield and purity. The ODN-peptides were further characterized by proteolysis with trypsin. Thermal denaturation studies with ssDNA targets showed little effect of the 5'-peptide modifications on the hybridization properties of the ODN. The effect of the nuclear signal peptides on antisense potency was evaluated in the freshwater ciliate Paramecium. A 3'-hexanol-modified 24-mer antisense ODN, complementary to the mRNA for calmodulin, alters regulation of membrane ion channels and swimming behavior of these cells. A 2'-O-methyl analog of this ODN was inactive, thus providing evidence that this activity in Paramecium is mediated by RNase H. Antisense ODN-nuclear signal peptide conjugates were transfected into the cells by electroporation. Surprisingly, these conjugates showed no antisense effects in comparison to a 5'-unmodified control ODN. Random peptides or amino acids conjugated to the 5'-terminus did not decrease antisense activity.

Women are at greater risk than men for malpositioning of the endotracheal tube after emergent intubation.

OBJECTIVES: To investigate the occurrence of endotracheal tube malpositioning after emergent intubation in critically ill adults and to determine the need for a routine postintubation chest radiography to assess endotracheal tube position. DESIGN: Prospective study. SETTING: All adult critical care and acute care units of a 560-bed university teaching hospital. PATIENTS: Study of 297 consecutive intubations (185 intubations in males and 112 intubations in females) in 238 adult patients. METHODS: Emergent endotracheal intubations were performed by resident physicians with supervision from an intensive care unit (ICU) or anesthesia attending physician or an anesthesia resident. After intubation, proper positioning of the endotracheal tube was verified by the intubating physician using clinical criteria, including auscultation of bilateral breath sounds, symmetric chest expansion, and palpation of the endotracheal tube cuff in the suprasternal notch. The endotracheal tube position relative to the lower anterior incisors or alveolar ridge was recorded using the centimeter markings printed on the endotracheal tube. A chest radiograph was obtained after intubation to verify endotracheal tube position. Appropriate endotracheal tube position on chest radiograph was defined as between > 2 and < or = 6 cm above the carina. MEASUREMENTS AND MAIN RESULTS: Of the 297 intubations, 26 were excluded from analysis because a chest radiograph was not obtained or the patient was not of normal stature. For the remaining 271 intubations, 42 (15.5%) endotracheal tubes were inappropriately placed, according to the radiographic assessment. The percentage of malpositioned endotracheal tubes was significantly higher in women than in men (61.9% vs. 38.1%, respectively; chi-square: p < .001). Thirty-three (78.6%) of 42 malpositioned endotracheal tubes were placed < 2 cm from the carina, with the highest occurrence (24/33) of proximal malposition occurring in women. Positioning of endotracheal tubes using the centimeter markings printed on the tube referenced to the lower incisors did not accurately identify malposition as documented by chest radiograph. CONCLUSIONS: Emergent endotracheal intubations result in a significant occurrence of malpositioned endotracheal tubes that are undetected by clinical evaluation. Malpositioning is not detected by routine clinical assessment, but only by chest radiograph. Women are at greater risk than men for endotracheal tube malpositioning after emergent intubation; in women, the endotracheal tube is more likely to be positioned too close to the carina. A chest radiograph for confirmation of endotracheal tube position after emergent intubation should remain the standard of practice.

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.

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.

Species-specific oligodeoxynucleotide probes for the identification of periodontal bacteria.

Oligodeoxynucleotide probes were developed for identification of the periodontal bacteria Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, B. intermedius types I and II, B. forsythus, Eikenella corrodens, Fusobacterium nucleatum, Haemophilus aphrophilus, Streptococcus intermedius, and Wolinella recta. Probes were designed by sequencing the 16S rRNA for each bacterium, identifying hypervariable regions, and chemically synthesizing species-specific probes. These probes were specific when tested against a panel of nucleic acids from closely related bacteria.

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.

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.

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.