Utility of risk-weighted surgical-pathological factors in early-stage cervical cancer.

BACKGROUND: Surgical-pathological risk factors were evaluated by weighting the magnitude of significance of multiple risk factors correlating to survival and treatment response in cervical cancer. METHODS: Multivariate analysis was performed for survival outcomes entering seven pathological factors obtained from 540 radical hysterectomy specimens in stage IA2-IIB cervical cancer cases. Hazard ratio (HR) in each risk factor was determined, and the sum of HR scores for the corresponding risk factors was determined per case. Survival curves and postoperative treatment response (concurrent chemoradiotherapy (CCRT) vs radiotherapy alone) were evaluated based on the extent of HR-weighted scores. RESULTS: Hazard ratios for risk factors relating to disease-free survival (DFS) was: lympho-vascular space invasion 3.95, nodal metastasis 3.88, adenocarcinoma 3.40, large tumour 2.36, positive margin 1.99, deep stromal invasion 1.29, and parametria invasion 1.21. The HR-weighted scoring method showed a high predictive value for recurrence (area-under-curve 0.836, P<0.001). Hazard ratio-weighted scores were negatively correlated to DFS, and the cases with score 12.5 showed 5-year DFS rate of 23.8%. Tumours with larger score offset the benefits of CCRT over radiotherapy alone for postoperative adjuvant treatment (P<0.001). CONCLUSION: Surgical-pathological risk factors provide valuable information for survival and management of early-stage cervical cancer when number and significance of risks are weighted.

A retrospective analysis of endometrial carcinoma cases surgically treated with or without para-aortic lymph node dissection followed by adjuvant chemotherapy.

PURPOSE: To analyze the efficacies of para-aortic node (PAN) dissection for patients undergoing surgery and adjuvant chemotherapy for endometrial carcinomas. METHODS: At the Osaka University Hospital and the Kaizuka City Hospital in Osaka, Japan, either pelvic lymph nodes (PLN) plus para-aortic lymph nodes (PAN) or PLN-only dissections were performed for endometrial carcinomas. An adjuvant chemotherapy using paclitaxel, epirubicin, and carboplatin was conducted for all such patients. A retrospective comparison of the efficacy of PAN dissection was conducted. RESULTS: Disease-free and overall survivals and frequency of PAN involvement at the first recurrence did not exhibit a statistically significant difference between the PLN-only group and the PLN + PAN group. Operation time was significantly longer in the PLN + PAN group than the PLN-only group, and the total blood loss was also significantly greater in the PLN+PAN group. CONCLUSION: PAN dissection may be omitted, without adverse effect on prognosis, for endometrial carcinoma patients with recurrence risks who undergo adjuvant chemotherapy using platinum, anthracycline and taxane derivatives.

Acute severe alcohol-induced bronchial asthma.

We report the case of a severe bronchial asthma attack 15 minutes after the ingestion of food containing small amounts of alcohol. Although an ethanol inhalation test was negative, an acetaldehyde inhalation test was positive. Furthermore, it was discovered that the patient was homozygous for a mutation of the aldehyde dehydrogenase-2 (ALDH-2) gene. We subsequently diagnosed his attack as acute severe alcohol-induced asthma. Since bronchial asthma patients who are homozygous for mutant ALDH-2 genes are susceptible to acute severe alcohol-induced asthma attacks, strict clinical attention is thought a necessity.

Stiffness of peripheral airway folding membrane in rabbits.

We have observed that small, membranous bronchioles from rabbits, in which the smooth muscle is not activated, experience a critical elastic buckling involving the whole airway wall during deflation of the lung. This implies that, at some point during the deflation, the airway wall goes from being in a state of tension to a state of compression. At the transition, there is neither net tension nor net compression in the wall, and the transmural pressure difference must, therefore, be zero. Thus at this point, the pressure difference across the muscle that results from the passive stress in the muscle is just balanced by the pressure difference across the folded mucosal membrane. We estimated the muscle stress, and hence the pressure across the muscle, from published data on rabbit trachealis (Opazo-Saez A and Paré PD, J Appl Physiol 77: 1638-1643, 1994) and equated this to the pressure across the folded membrane. By using a theoretical prediction of this pressure (Lambert RK, Codd SL, Alley MR, and Pack RJ, J Appl Physiol 77: 1206-1216, 1994), together with the results of our morphometric measurements on these airways, we estimated that the flexural rigidity of the folding membrane in peripheral rabbit airways is of the order of 10(-12) Pa x m3. This value implies that, in these airways, membrane folding provides significant resistance to airway smooth muscle shortening.

Compliance of peripheral airways deduced from morphometry.

Insights into airway mechanics were sought by applying morphometric techniques to rabbit lungs fixed at several lung recoil pressures. Rabbits were treated with either nebulized carbachol followed by iv administration of carbachol or with saline solution (sham). The lungs were held at one of six values of positive end-expiratory pressure (PEEP; 10, 7, 4, 2, 0, and -4 cmH(2)O) while the animal was killed and formalin was circulated through the lungs. The lungs were removed and left in a bath of formalin for 24 h. Standard airway morphometric measurements were made on membranous bronchiole slices taken from representative blocks of tissue. Reductions in PEEP produced the expected reductions in lumen area in the carbachol-treated airways but not in the sham-treated airways for PEEP > 2 cmH(2)O. Sham-treated airways remained more open than expected until they collapsed into an oval shape at PEEPs between 4 and 2 cmH(2)O. The carbachol-treated airways exhibited this behavior at PEEP = -4 cmH(2)O. The smallest airways, which had relatively thicker walls, collapsed less than larger airways. We postulate that this behavior implies that peribronchial stress is greater than lumen pressure on collapse into the oval shape. Resistance to buckling increases with the thickness-to-radius ratio of the airway wall, which explains why the smallest airways are the most open. The development of epithelial folds appeared to follow the theoretical prediction of a previous study (Lambert RK, Codd SL, Alley MR, and Pack RJ. J Appl Physiol 77: 1206-1216, 1994).

l-Menthol-induced [Ca2+]i increase and impulses in cultured sensory neurons.

We investigated the effects of l-menthol on cultured dorsal root ganglion (DRG) cells, instead of free nerve endings of sensory fibers. Using Fura-2 microfluorimetry, we identified a few DRG neurons that showed an increase in intracellular free Ca2+ concentration ([Ca2+]i) in response to l-menthol. They made up only 10% of the neurons activated by a high K+ solution. l-Menthol induced the [Ca2+]i increase in a dose-dependent manner, with an EC50 of 37.9 microM and a Hill coefficient of 0.97. A related compound, cyclohexanol, had no effect. When extracellular Ca2+ was removed, l-menthol did not induce the [Ca2+]i increase. Whole-cell current-clamp recordings revealed that l-menthol induced depolarization (13.2 mV, receptor potential) leading to impulses. We conclude that l-menthol induced the impulses through activation of menthol receptors in a small subset of the cultured sensory neurons.

Mechanical properties of the tracheal mucosal membrane in the rabbit. I. steady-state stiffness as a function of age.

Airway responsiveness is exaggerated in infancy and declines with maturation. These age-related differences (R.S. Tepper, T. Du, A. Styhler, M. Ludwig, and J.G. Martin. Am. J. Respir. Crit. Care Med. 151: 836-840, 1995; R.S. Tepper, S.J. Gunst, C.M. Doerschuk, Y. Shen, and W. Bray. J. Appl. Physiol. 78: 505-512, 1995; R.S. Tepper, J. Stevens, and H. Eigen. Am. J. Respir. Crit. Care Med. 149: 678-681, 1994) could be due to changes in the smooth muscle, the lung, and/or the airway wall. Folding of the mucosal membrane can provide an elastic load (R.K. Lambert, J. Appl. Physiol. 71: 666-673, 1991), which impedes smooth muscle shortening. We hypothesized that increased stiffness of the mucosal membrane occurs during aging, causing an increased mechanical load on airway smooth muscle and a decrease in airway responsiveness. Forty female New Zealand White rabbits between 0.75 and 35 mo of age were studied. Rectangular mucosal membrane strips oriented both longitudinally and circumferentially to the long axis of the trachea were dissected, and the stress-strain relationships of each strip were tested. The results showed that the membrane was stiffer in the longitudinal than in the circumferential direction of the airway. However, there was no significant change with age in either orientation. We conclude that the mechanical properties of the airway mucosal membrane did not change during maturation and were not likely to influence age-related changes in airway responsiveness.

Mechanical properties of the tracheal mucosal membrane in the rabbit. II. Morphometric analysis.

Folding of the airway mucosal membrane provides a mechanical load that impedes airway smooth muscle contraction. Mechanical testing of rabbit tracheal mucosal membrane showed that the membrane is stiffer in the longitudinal than in the circumferential direction of the airway. To explain this difference in the mechanical properties, we studied the morphological structure of the rabbit tracheal mucosal membrane in both longitudinal and circumferential directions. The collagen fibers were found to form a random meshwork, which would not account for differences in stiffness in the longitudinal and circumferential directions. The volume fraction of the elastic fibers was measured using a point-counting technique. The orientation of the elastic fibers in the tissue samples was measured using a new method based on simple geometry and probability. The results showed that the volume fraction of the elastic fibers in the rabbit tracheal mucosal membrane was approximately 5% and that the elastic fibers were mainly oriented in the longitudinal direction. Age had no statistically significant effect on either the volume fraction or the orientation of the elastic fibers. Linear correlations were found between the steady-state stiffness and the quantity of the elastic fibers oriented in the direction of testing.

[A study of the long-term effectiveness of an outpatient pulmonary rehabilitation program].

Pulmonary rehabilitation is one of the most important components of comprehensive care for patients with significant disability due to chronic respiratory failure. Because pulmonary rehabilitation has not been popular in Japan, the long-term effectiveness of pulmonary rehabilitation has rarely been reported. We therefore examined the long-term effectiveness of an outpatient pulmonary rehabilitation program for patients with chronic respiratory failure. Our program was composed of a once-a-week introduction program for 2 months and a support program that was continued every 4 weeks as long as possible. Thirty stable patients with chronic respiratory failure were enrolled in the program; 21 patients (COPD: 15, lung complications of tuberculosis: 6) completed the 9-week introduction program and the ensuing 6-month support program. Good compliance with the home training regimen was maintained during the period. The introduction program significantly alleviated dyspnea (Fletcher's grade: 3.3 to 3.0, p < 0.01) and improved the data for activity (Spector's score: 5.3 to 5.8, p < 0.01) and 6-minute walking distance (319 to 384 m, p < 0.01). These benefits were sustained during the 6-month support program. We concluded that outpatient pulmonary rehabilitation can alleviate dyspnea and improve the activity and exercise tolerance of patients with chronic respiratory failure, and that the effectiveness of training can be well maintained with a minimal support program.

Ca2+ entry channels in rat thoracic aortic smooth muscle cells activated by endothelin-1.

The contraction of the rat aorta induced by endothelin-1 (ET-1) requires entry of extracellular Ca2+, but involvement of voltage-operated Ca2+ channel is minor. Using whole-cell recordings of patch-clamp and monitoring of the intracellular free Ca2+ concentration ([Ca2+]i), we characterized Ca2+ entry channels in A7r5 cells activated by ET-1. ET-1 activates three types of voltage-independent Ca2+ entry channels: two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC). Furthermore, it was found that these channels can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. NSCC-1 is resistant to SK&F 96365, but sensitive to LOE 908, whereas NSCC-2 is sensitive to both SK&F 96365 and LOE 908. SOCC is sensitive to SK&F 96365, but resistant to LOE 908. Using these channel blockers, we analyzed Ca2+ entry channels involved in the ET-1-induced contractions of rat thoracic aorta and increases in [Ca2+]i of single smooth muscle cells. The responses to lower concentrations of ET-1 (< or = 0.1 nM) were abolished by either SK&F 96365 or LOE 908 alone. In contrast, the responses to higher concentrations of ET-1 (> or = 1 nM) were suppressed by SK&F 96365 or LOE 908 to about 10% and 35% of controls, respectively, and abolished by combined treatment with SK&F 96365 and LOE 908. These results show that the responses of rat aorta to lower concentrations of ET-1 involve only one Ca2+ channel that is sensitive to SK&F 96365 and LOE 908 (NSCC-2), whereas those to higher concentrations of ET-1 involve NSCC-1, NSCC-2 and SOCC, contributing 10%, 55% and 35%, respectively, to total Ca2+ entry.

Pharmacological characterization of Ca2+ entry channels in endothelin-1-induced contraction of rat aorta using LOE 908 and SK&F 96365.

We have recently shown that endothelin-1 (ET-1) activates two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and store-operated Ca2+ channel (SOCC). These channels can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. Here we characterized Ca2+ entry channels involved in ET-1-induced contractions of rat thoracic aortic rings and increases in the intracellular free Ca2+ concentration ([Ca2+]i) of single smooth muscle cells using these blockers. LOE 908 or a blocker of voltage-operated Ca2+ channel nifedipine had no effect on the contractions and increases in [Ca2+]i induced by thapsigargin or ionomycin, whereas SK&F 96365 abolished them. The contractions and increases in [Ca2+]i induced by ET-1 depended on extracellular Ca2+ but were resistant to nifedipine. The responses to lower concentrations (< or =0.1 nM) of ET-1 were abolished by either SK&F 96365 or LOE 908. The responses to higher concentrations (> or = 1 nM) were abolished by SK&F 96365, but were partially resistant to LOE 908. SK&F 96365 inhibited the LOE 908-resistant contractions induced by higher concentrations of ET-1 with IC50 values similar to those for contractions induced by thapsigargin or ionomycin. These results show that the contractions and increases in [Ca2+]i of rat aortic smooth muscles at lower concentrations of ET-1 involve only one Ca2+ entry channel which is sensitive to SK&F 96365 and LOE 908 (NSCC-2), whereas those at higher concentrations of ET-1 involve another Ca2+ entry channel which is sensitive to SK&F 96365 but resistant to LOE 908 (SOCC) in addition to the former channel.

Activation of three types of voltage-independent Ca2+ channel in A7r5 cells by endothelin-1 as revealed by a novel Ca2+ channel blocker LOE 908.

1. We have shown that in addition to voltage-operated Ca2+ channel (VOC), endothelin-1 (ET-1) activates two types of Ca2+-permeable nonselective cation channel (NSCC) in A7r5 cells: its lower concentrations (< or = 1 nM; lower [ET-1]) activate only an SK&F 96365-resistant channel (NSCC-1), whereas its higher concentrations (> or = 10 nM; higher [ET-1]) activate an SK&F 96365-sensitive channel (NSCC-2) as well. 2. We now characterized the effects of a blocker of Ca2+ entry channel LOE 908 on NSCCs and store-operated Ca2+ channel (SOCC) in A7r5 cells, and using two drugs, clarified the involvement of these channels in the ET-1-induced increase in the intracellular free Ca2+ concentrations ([Ca2+]i). Whole-cell recordings and [Ca2+]i monitoring with fluo-3 were used. 3. LOE 908 up to 10 microM had no effect on increases in [Ca2+]i induced by thapsigargin or ionomycin, but SK&F 96365 abolished them. 4. In the cells clamped at -60 mV, both lower and higher [ET-1] induced inward currents with linear iv relationships and the reversal potentials of -15.0 mV. Thapsigargin induced no currents. 5. In the presence of nifedipine, lower [ET-1] induced a sustained increase in [Ca2+]i, whereas higher [ET-1] induced a transient peak and a sustained increase. The sustained increases by lower and higher [ET-1] were abolished by removal of extracellular Ca2+, and they were suppressed by LOE 908 to 0 and 35%, respectively, with the LOE 908-resistant part being abolished by SK&F 96365. 6. These results show that LOE 908 is a blocker of NSCCs without effect on SOCC, and that the increase in [Ca2+]i at lower [ET-1] results from Ca2+ entry through NSCC-1 in addition to VOC, whereas the increase at higher [ET-1] involves NSCC-1, NSCC-2 and SOCC in addition to VOC.

Mechanical properties of lung parenchyma during bronchoconstriction.

Interdependence between airways and the lung parenchyma is thought to be a major mechanism preventing excessive airway narrowing during bronchoconstriction. Because the elastance of the lung increases during bronchoconstriction, the lung's tethering force could also increase, further attenuating bronchoconstriction. We hypothesized that the bulk (kappa) and shear moduli (mu) of the lung increase similarly during bronchoconstriction. To test this hypothesis, we excised rabbit lungs and measured the lung volume, pulmonary elastance, kappa, and mu at transpulmonary pressures of 4, 6, 8, 12, and 16 cmH2O using pressure-volume curves, slow oscillations of the lung, and an indentation test. Bronchoconstriction was induced by nebulizing carbachol by using small tidal-volume ventilation to prevent hyperinflation. The measurement of kappa and mu was repeated after carbachol treatment. After carbachol treatment, the increase in kappa was significantly greater than that in mu. The estimated value for mu was approximately 0.5 x transpulmonary pressure both before and after carbachol treatment. These data suggest that the tethering effect of the lung parenchyma, which serves to attenuate bronchoconstriction, is not significantly increased during carbachol administration unless there is hyperinflation.

Endothelin-induced apoptosis of A375 human melanoma cells.

Endothelin-1 (ET-1) inhibited serum-dependent growth of asynchronized A375 human melanoma cells, and the growth inhibitory effect was markedly enhanced when ET-1 was applied to the cells synchronized at G1/S boundary by double thymidine blocks. Flow cytometric analysis revealed that ET-1 did not inhibit the cell cycle progression after the release of the block but caused a significant increase of the hypodiploid cell population that is characteristic of apoptotic cell death. ET-1-induced apoptosis was confirmed by the appearance of chromatin condensation on nuclear staining and DNA fragmentation on gel electrophoresis. The increase in the hypodiploid cell peak was manifest within 16 h of exposure to 5 nM ET-1. Within the same time range, ET-1 caused actin reorganization and drastic morphological changes of the surviving cells from epithelioid to an elongated bipolar shape. These phenotypical changes were preceded by ET-1-induced increase and nuclear accumulation of the tumor suppressor protein p53. All of these effects of ET-1 were mediated by ETB via a pertussis toxin-sensitive G protein. Flow cytometric analysis with fluorescent dye-labeled ET-1 revealed up-regulation of ETB expressed by the cells in G1/early S phases, and overexpression of the receptor protein by cDNA microinjection conferred the responsiveness (both apoptosis and morphological changes) to ET-1 irrespective of the position of the cell in the cell cycle. These results indicated the presence of ETB-mediated signaling pathways to apoptotic cell machinery and cytoskeletal organization. Furthermore, the densities of ETB expressed by individual A375 melanoma cells appeared to be regulated by a cell cycle-dependent mechanism, and the receptor density can be a limiting factor to control the apoptotic and cytoskeletal responses of the cells to ET-1. Although the molecular mechanisms remain to be elucidated, these findings added a new dimension to the diverse biological activities of ETs and also indicated a novel mechanism to control the responsiveness of the cell to the peptides.

Functional changes in aging polymorphonuclear leukocytes.

BACKGROUND: Previous studies from our laboratory have shown that the expression of L-selectin on polymorphonuclear neutrophils (PMN) decreases as the cell ages in the circulation and that these older PMN have more fragmented DNA and show morphological features of apoptosis. METHODS AND RESULTS: The present study was designed to compare the functional capabilities of PMN expressing low levels of L-selectin (L-selectin[low]) and the total population of PMN they were isolated from (L-selectin[mixed]). The results show no difference of the baseline filamentous actin (F-actin) content between PMN expressing low and high levels of L-selectin. However, the ability of L-selectin(low) PMN to assemble F-actin was impaired after stimulation by n-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (1 nmol/L fMLP: P<.02, 10 nmol/L fMLP: P<.01). The ability of L-selectin(low) PMN to change shape when stimulated (10 nmol/L fMLP) was also decreased (P<.05). Filtration studies showed no difference in baseline deformability between L-selectin(low) and L-selectin(mixed) leukocytes, but the L-selectin(low) cells showed a decreased ability to stiffen after fMLP stimulation (P<.05). L-selectin(low) cells demonstrated a decreased ability to migrate toward a chemoattractant (1, 3, and 10 nmol/L fMLP) (P<.004) but have an enhanced ability to upregulate CD18 (P<.00002) and produce hydrogen peroxide (P<.00004). CONCLUSIONS: We conclude that PMN undergo substantial functional changes as they age in the circulation.

Eosinophilic leukocyte accumulation during vagally induced bronchoconstriction.

Eosinophilic leukocytes (eosinophils) are important effector cells in allergic inflammatory diseases such as asthma, in which significant accumulation of these cells is observed in the bronchial mucosa. However, there is little information about the relationships between bronchoconstriction and accumulation of eosinophils. We hypothesized that eosinophils are retained in the bronchial vasculature in the inner airway wall during bronchoconstriction because of deformation of the mucosal membrane. To test this hypothesis we induced unilateral bronchoconstriction in open chest guinea pigs by stimulating the right vagus nerve and compared the accumulation of eosinophils in the airway wall of the constricted and contralateral unconstricted lungs using histologic specimens. Results show that the density of eosinophils (number of cells/wall area) significantly increased in the inner wall and decreased in the adventitia of the constricted airways compared with the contralateral unconstricted airways. There was a positive relationship between the amount of smooth muscle shortening and the eosinophil density in the inner wall. On the other hand, this relationship was significantly negative in the adventitia. Atropine completely inhibited the eosinophil accumulation in the inner wall. These data suggest that eosinophils can accumulate in the airway inner wall during bronchoconstriction because of geometrical factors.

Bromodeoxyuridine-induced expression of endothelin A in A375 human melanoma cells.

Expression of endothelin (ET) receptor subtypes was examined in an experimental model of A375 human melanoma cell differentiation using the pyrimidine analog bromodeoxyuridine (BUdR). BUdR (10 microM)-treated cells had an increased surface area and an increased dendricity, were contact-inhibited and lacked tumorigenecity in athymic nude mice. The untreated A375 cells exclusively expressed ETB and BUdR-induced phenotypical changes were accompanied by induction of ETA expression as evidenced by northern blotting, [125I]ET-1 binding assay and [Ca2+]i measurement. Thus, BUdR-induced differentiation of A375 melanoma cells may provide a model system to study the receptor subtype switch in melanocyte development.

Fast Fourier transform analysis of dynamic data: sine wave stress-strain analysis of biological tissue.

We propose an improvement to the dynamic oscillation method using as an example the measurement of the tensile stiffness of the rabbit tracheal mucosal membrane. A sine wave oscillation technique was used to study the tissue mechanical properties. A mathematical model was developed using fast Fourier analysis. After mathematically eliminating the machine response, this analysis reveals the tissue frequency response over a wide range of frequencies. This study addresses the advantages of using Fourier analysis to interpret dynamic properties of biological tissue and provides a complete description of how to obtain the pure tissue response.

Human airway narrowing measured using high resolution computed tomography.

Exaggerated airway narrowing in response to bronchoconstricting stimuli is a characteristic feature of asthmatic subjects. It is unknown whether the site of airway narrowing differs in asthmatic subjects from that observed in normal subjects. Increased airway wall thickness has been suggested as a contributing cause for airway hyperresponsiveness in asthma, based on histologic measurements. We measured airway wall thickness and the site and magnitude of airway narrowing in response to inhaled methacholine in normal subjects and in patients with mild to moderate asthma using high resolution computed tomography (HRCT). After a comparable decrease in FEV1, there were no differences in the site or magnitude of airway narrowing for any category of airway size in asthmatic subjects and normals. However, the results show that the smaller airways of the asthmatic subjects are significantly thickened and that the airway wall area does not change after bronchoconstriction whereas it decreases in normal subjects. We conclude that airway wall thickening and the lack of a change in airway wall dimensions following bronchoconstricting stimuli could contribute to exaggerated airway narrowing in asthma.