Microsatellite alterations in plasma DNA of small cell lung cancer patients.

Microsatellite instability is an important characteristic of many tumor types especially those associated with hereditary non-polyposis colorectal carcinoma (HNPCC) syndrome. Microsatellite alterations in 50% of primary small cell lung carcinoma (SCLC) have been found. These alterations were also found in the sputum. Because neoplastic characteristics such as decreased strand stability9 and ras mutations have been found in the plasma DNA of cancer patients, we looked for microsatellite alterations in the plasma of SCLC patients. A microsatellite alteration was present in 16 out of 21 (76%) SCLC tumors and in 15 out of 21 (71%) plasma samples. In one case, the alteration was present only in the plasma DNA. If confirmed in larger studies, microsatellite analysis of plasma DNA might constitute a new tool for tumor staging, management and, possibly, detection.

The nature of antioxidant defense mechanisms: a lesson from transgenic studies.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of many clinical disorders such as adult respiratory distress syndrome, ischemia-reperfusion injury, atherosclerosis, neurodegenerative diseases, and cancer. Genetically engineered animal models have been used as a tool for understanding the function of various antioxidant enzymes in cellular defense mechanisms against various types of oxidant tissue injury. Transgenic mice overexpressing three isoforms of superoxide dismutase, catalase, and the cellular glutathione peroxidase (GSHPx-1) in various tissues show an increased tolerance to ischemia-reperfusion heart and brain injury, hyperoxia, cold-induced brain edema, adriamycin, and paraquat toxicity. These results have provided for the first time direct evidence demonstrating the importance of each of these antioxidant enzymes in protecting the animals against the injury resulting from these insults, as well as the effect of an enhanced level of antioxidant in ameliorating the oxidant tissue injury. To evaluate further the nature of these enzymes in antioxidant defense, gene knockout mice deficient in copper-zinc superoxide dismutase (CuZnSOD) and GSHPx-1 have also been generated in our laboratory. These mice developed normally and showed no marked pathologic changes under normal physiologic conditions. In addition, a deficiency in these genes had no effects on animal survival under hyperoxida. However, these knockout mice exhibited a pronounced susceptibility to paraquat toxicity and myocardial ischemia-reperfusion injury. Furthermore, female mice lacking CuZnSOD also displayed a marked increase in postimplantation embryonic lethality. These animals should provide a useful model for uncovering the identity of ROS that participate in the pathogenesis of various clinical disorders and for defining the role of each antioxidant enzyme in cellular defense against oxidant-mediated tissue injury.

Aggressive intensive care treatment of very elderly patients with tetanus is justified.

Tetanus is now rare in industrialized countries, occurring mainly in elderly patients. To assess whether aggressive therapy of these patients in the intensive care unit is justified, we retrospectively studied all patients with tetanus hospitalized in our institution between 1968 and 1989. Patients over the age of 70 years fared as well as those under 70 years and recovered without sequelae. These results favor aggressive treatment of elderly patients with tetanus in the intensive care unit.

[Episodic laryngeal dyskinesia: a functional cause of stridor]. / La dyskinésie laryngée épisodique: une cause fonctionnelle de stridor.

We present five cases of episodic laryngeal dyskinesia. This term describes an entity associating acute dyspnea with inspiratory and/or expiratory stridor, sometimes ending in acute respiratory distress. The origin of this trouble comes from paradoxal contraction of the vocal cords, which is reversible and recurrent. The clinical presentation presents as a differential diagnosis for serious disorders such as bronchial asthma, laryngeal oedema, or stenosing lesions of the upper airways. The definitive diagnosis is made by excluding organic pathology, by visualising laryngeal spasm and by the inspection of flow volume curve. A comparison of all the published papers since 1974 reveals the benign nature of this "noisy" disorder and to bring out the diagnostic criteria thus avoiding too aggressive an approach in these patients.

Mode of presentation and diagnosis of bacterial pneumonia in human immunodeficiency virus-infected patients.

Bacterial pneumonia (BP) has recently been reported to be more frequent in human immunodeficiency virus (HIV)-infected patients than in normal hosts. This study reviews the clinical and radiologic manifestations of BP in 132 consecutive pulmonary episodes over a 15-month period. BP was defined on a clinical basis as a pulmonary infiltrate accompanied by fever and improving in a few days with conventional antibiotics (trimethoprim-sulfamethoxazole excluded). In patients undergoing bronchoscopy (97 procedures), semiquantitative cultures and cell differentials of bronchoalveolar lavage (BAL) were performed, in addition to conventional staining and cultures for opportunistic infections. BP were frequent (45%), and the usual community-acquired pathogens were found. The radiologic manifestations of BP were often unusual, however, and 47% were indistinguishable from the typical appearance of Pneumocystis carinii pneumonia. BAL cultures had a sensitivity of 83 or 23%, depending on whether antibiotics were administered before bronchoscopy, using a cutoff value of greater than or equal to 10(4) bacteria/ml. The specificity of BAL culture was of 80.5% if patients with P. carinii pneumonia were taken as a control group. We conclude that BP is frequently encountered in HIV-infected patients. The clinical and radiologic presentation of BP may be indistinguishable from that of opportunistic infections. Semiquantitative cultures of BAL appear a valuable diagnostic tool to avoid unnecessary invasive diagnostic procedures or treatments.

Mice deficient in cellular glutathione peroxidase develop normally and show no increased sensitivity to hyperoxia.

Glutathione peroxidase, a selenium-containing enzyme, is believed to protect cells from the toxicity of hydroperoxides. The physiological role of this enzyme has previously been implicated mainly using animals fed with a selenium-deficient diet. Although selenium deficiency also affects the activity of several other cellular selenium-containing enzymes, a dramatic decrease of glutathione peroxidase activity has been postulated to play a role in the pathogenesis of a number of diseases, particularly those whose progression is associated with an overproduction of reactive oxygen species, found in selenium-deficient animals. To further clarify the physiological relevance of this enzyme, a model of mice deficient in cellular glutathione peroxidase (GSHPx-1), the major isoform of glutathione peroxidase ubiquitously expressed in all types of cells, was generated by gene-targeting technology. Mice deficient in this enzyme were apparently healthy and fertile and showed no increased sensitivity to hyperoxia. Their tissues exhibited neither a retarded rate in consuming extracellular hydrogen peroxide nor an increased content of protein carbonyl groups and lipid peroxidation compared with those of wild-type mice. However, platelets from GSHPx-1-deficient mice incubated with arachidonic acid generated less 12-hydroxyeicosatetraenoic acid and more polar products relative to control platelets at a higher concentration of arachidonic acid, presumably reflecting a decreased ability to reduce the 12-hydroperoxyeicosatetraenoic acid intermediate. These results suggest that the contribution of GSHPx-1 to the cellular antioxidant mechanism under normal animal development and physiological conditions and to the pulmonary defense against hyperoxic insult is very limited. Nevertheless, the potential antioxidant role of this enzyme in protecting cells and animals against the pathogenic effect of reactive oxygen species in other disorders remains to be defined. The knockout mouse model described in this report will also provide a new tool for future study to distinguish the physiological role of this enzyme from other selenium-containing proteins in mammals under normal and disease states.

Variation in cellular glutathione peroxidase activity in lens epithelial cells, transgenics and knockouts does not significantly change the response to H2O2 stress.

This investigation examines the contribution of glutathione peroxidase (GSHPx-1) in degrading H2O2 in lens preparations. Rabbit (N/N1003A) and normal and GSHPx-1 transfected mouse (alpha TN4-1) lens epithelial cell lines and normal and GSHPx-1 transgenic and knockout mouse lenses were utilized. GSHPx-1 activity in the cell lines was increased from two-fold to about four-fold, in the lenses from transgenics more than four-fold and the lenses from knockouts had less than 3% of normal GSHPx-1 activity. The transgenic and knockout mice as well as their lenses appeared normal for up to 3 to 4 months, the longest period of observation. The preparations were subjected to oxidative stress by placing them either in a medium containing 120 or 300 microM H2O2 or utilizing photochemical stress where the H2O2 levels normally rise to about 100 microM over a few hours in the presence of a normal lens. With all preparations, it was found that either markedly increasing or eliminating GSHPx-1 activity had only a small effect on the system's ability to metabolize H2O2, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of GSSG reductase (GSSG Red) and 3-aminotriazole (3-AT), an inhibitor of catalase, also had little effect. However, the addition of both inhibitors caused a marked decrease in H2O2 degradation. Examination of the distribution of GSHPx-1 in the lens indicated that the activity per milligram of protein was evenly distributed between the epithelium and the remainder of the lens in the normal lens and was about 1.7-fold greater in the epithelium of transgenic lenses than in the remainder of the lens. Surprisingly, the distribution of GSSG Red was quite different with eight- to ten-fold more activity in the epithelium. Catalase was also found to be concentrated in the epithelium. With H2O2 exposure, a rapid loss of non-protein thiol (NP-thiol) was found in cell cultures and in the epithelia of cultured lenses. However, the remainder of the lens showed little change in NP-thiol. The variation of GSHPx-1 activity did not influence the NP-thiol changes which occurred more rapidly and to a greater extent in the presence of BCNU. The addition of BCNU also caused a decrease in total lens NP-thiol. Examination of thymidine incorporation and choline transport, indicators of nuclear and membrane function, also reflects the H2O2 degradation data, showing little difference in the degree to which H2O2 effects these parameters in lenses from normal and transgenic animals. Catalase activity is four- to six-fold greater than GSHPX-1 activity in the alpha TN4-1 cell lines, about three-fold lower in the rabbit cell line and, remarkably, about 18-fold lower than the peroxidase in the normal mouse lens. In spite of such observations, the consistent overall conclusion is that GSHPx-1 and catalase function together but when GSHPx-1 is knocked out or GSSG Red is inhibited, catalase is able to protect the system from H2O2 stress. Indeed, the young mouse does not appear to require GSH Px-1 for normal function.

Activation of human macrophages by mechanical ventilation in vitro.

Positive-pressure mechanical ventilation supports gas exchange in patients with respiratory failure but is also responsible for significant lung injury. In this study, we have developed an in vitro model in which isolated lung cells can be submitted to a prolonged cyclic pressure-stretching strain resembling that of conventional mechanical ventilation. In this model, cells cultured on a Silastic membrane were elongated up to 7% of their initial diameter, corresponding to a 12% increase in cell surface. The lung macrophage was identified as the main cellular source for critical inflammatory mediators such as tumor necrosis factor-alpha, the chemokines interleukin (IL)-8 and -6, and matrix metalloproteinase-9 in this model system of mechanical ventilation. These mediators were measured in supernatants from ventilated alveolar macrophages, monocyte-derived macrophages, and promonocytic THP-1 cells. Nuclear factor-kappaB was found to be activated in ventilated macrophages. Synergistic proinflammatory effects of mechanical stress and molecules such as bacterial endotoxin were observed, suggesting that mechanical ventilation might be particularly deleterious in preinjured or infected lungs. Dexamethasone prevented IL-8 and tumor necrosis factor-alpha secretion in ventilated macrophages. Mechanical ventilation induced low levels of IL-8 secretion by alveolar type II-like cells. Other lung cell types such as endothelial cells, bronchial cells, and fibroblasts failed to produce IL-8 in response to a prolonged cyclic pressure-stretching load. This model is of particular value for exploring physical stress-induced signaling pathways, as well as for testing the effects of novel ventilatory strategies or adjunctive substances aimed at modulating cell activation induced by mechanical ventilation.

Peroxide-induced damage in lenses of transgenic mice with deficient and elevated levels of glutathione peroxidase.

Transgenic mice with elevated glutathione peroxidase (GSHPx) activity and gene knockout animals with a deficiency of the enzyme were used to investigate the role of GSHPx in defending the lens against H2O2-induced damage. The effects of peroxide on cultured lenses were determined by using light and transmission electron microscopy to evaluate morphological changes occurring in the epithelium and superficial cortex of the central and equatorial regions of the lens. DNA single-strand breaks in the epithelium were also examined. Following a 30-min exposure to 25 microM H2O2, lenses from normal animals showed distinct changes in the morphology of both the epithelium and superficial cortex. The damage to these cells was extensive in lenses of gene knockout mice in which activity of GSHPx was undetectable. In marked contrast, lenses of transgenic mice, which had 5-fold higher activities of GSHPx, were able to resist the cytotoxic effects. Similar to damage to cell morphology, the extent of DNA strand breaks was significantly lower (40% of control) in H2O2-exposed lenses as compared to normal lenses while DNA damage in gene knockout lenses was 5 times greater than that of GSHPx-rich transgenic lenses. The present studies extend our previous findings on the role of the glutathione redox cycle in the detoxification of peroxide and demonstrate that an increase in GSHPx activity protects the lens against peroxide-induced changes in cell morphology and DNA strand breaks.

Glutathione peroxidase knockout mice are susceptible to myocardial ischemia reperfusion injury.

BACKGROUND: To test our hypothesis that intracellular antioxidant enzymes constitute a cellular defense against acute stress, we studied myocardial ischemia reperfusion injury in the setting of reduced level of glutathione peroxidase using GSHPx-1 gene knockout mice. METHODS: Knockout mice were developed by disrupting the coding sequence of GSHPx-1 gene after inserting a neomycin resistance gene derived from pMCIpol A into the EcoRI site located in exon 2. Isolated perfused hearts were prepared from two groups of mice-knockout and nontransgenic controls. A 4-0 silk was attached to the apex of the heart which in turn was attached to a force transducer. Hearts were perfused by the Langendorff mode, and after 20 minutes of stabilization subjected to 30 minutes of ischemia followed by 2 hours of reperfusion. The force developed by the heart (DF) and the first derivative of DF (dF/dt) were recorded. Creatine kinase (CK) release was measured in the perfusate and the infarct size was measured at the end of each experiment. RESULTS: For both GSHPx-1 knockout and nontransgenic control groups, DF and dF/dt were significantly lower during early postischemic reperfusion compared with baseline, but these values were significantly higher for the control group than the knockout mice throughout most of the reperfusion period. CK release from the heart increased during reperfusion for both groups, but this increase was significantly lower for the control group. The infarct size was also smaller for the control mice as compared with knockouts. CONCLUSIONS: The results indicate that the knockout mice are more susceptible to ischemia reperfusion injury, suggesting the importance of GSHPx-1 gene in myocardial protection from ischemic reperfusion injury.