Mutations in the LKB1 tumour suppressor are frequently detected in tumours from Caucasian but not Asian lung cancer patients.

Somatic mutations of LKB1 tumour suppressor gene have been detected in human cancers including non-small cell lung cancer (NSCLC). The relationship between LKB1 mutations and clinicopathological characteristics and other common oncogene mutations in NSCLC is inadequately described. In this study we evaluated tumour specimens from 310 patients with NSCLC including those with adenocarcinoma, adenosquamous carcinoma, and squamous cell carcinoma histologies. Tumours were obtained from patients of US (n=143) and Korean (n=167) origin and screened for LKB1, KRAS, BRAF, and EGFR mutations using RT-PCR-based SURVEYOR-WAVE method followed by Sanger sequencing. We detected mutations in the LKB1 gene in 34 tumours (11%). LKB1 mutation frequency was higher in NSCLC tumours of US origin (17%) compared with 5% in NSCLCs of Korean origin (P=0.001). They tended to occur more commonly in adenocarcinomas (13%) than in squamous cell carcinomas (5%) (P=0.066). LKB1 mutations associated with smoking history (P=0.007) and KRAS mutations (P=0.042) were almost mutually exclusive with EGFR mutations (P=0.002). The outcome of stages I and II NSCLC patients treated with surgery alone did not significantly differ based on LKB1 mutation status. Our study provides clinical and molecular characteristics of NSCLC, which harbour LKB1 mutations.

NOS and COX isoforms and abnormal microvessel responses to CO2 and H+ in hyperoxia-injured lungs.

The aim of the present study was to compare microvessel responses to hypercapnic and isocapnic acidosis in hyperoxia-injured lungs and to assess the role of constitutive and inducible forms of nitric oxide synthase (NOS) and cyclo-oxygenase (COX). Real-time confocal luminescence microscopy was used to measure changes in the diameter of acinar arterioles, venules and capillaries in response to stimulation with hypercapnic and isocapnic acidosis in isolated rat lungs injured by 90% oxygen exposure for 48 h. Observations were made with and without inhibition of constitutive (endothelial constitutive NOS (ecNOS) and COX-1) and inducible isoforms (iNOS and COX-2) of NOS and COX. Upregulation of NOS was assessed by measuring enzyme levels in lung homogenates by Western blot analysis and enhancement of the COX-related pathway was judged from perfusate concentrations of 6-ketoprostaglandin F1alpha. ecNOS and COX-1, but not iNOS and COX-2, were upregulated in hyperoxia-injured lungs. The nitric oxide produced by ecNOS attenuated COX-1 activity in injured arterioles and venules, but carbon dioxide enhanced it, leading to paradoxical dilatation of these microvessels under hypercapnic conditions with ecNOS inhibition. Although a high hydrogen ion concentration was unnecessary for excitation of COX-1, venule constriction in response to H+ was enhanced by COX-1 inhibition. Constitutive, but not inducible, isoforms of cyclo-oxygenase and nitric oxide synthase play an important role in abnormal microvessel responses to carbon dioxide and hydrogen ions in hyperoxia-injured lungs.

Effects of hypercapnia and hypocapnia on [Ca2+]i mobilization in human pulmonary artery endothelial cells.

The hydrogen ion is an important factor in the alteration of vascular tone in pulmonary circulation. Endothelial cells modulate vascular tone by producing vasoactive substances such as prostacyclin (PGI2) through a process depending on intracellular Ca2+ concentration ([Ca2+]i). We studied the influence of CO2-related pH changes on [Ca2+]i and PGI2 production in human pulmonary artery endothelial cells (HPAECs). Hypercapnic acidosis appreciably increased [Ca2+]i from 112 +/- 24 to 157 +/- 38 nmol/l. Intracellular acidification at a normal extracellular pH increased [Ca2+]i comparable to that observed during hypercapnic acidosis. The hypercapnia-induced increase in [Ca2+]i was unchanged by the removal of Ca2+ from the extracellular medium or by the depletion of thapsigargin-sensitive intracellular Ca2+ stores. Hypercapnic acidosis may thus release Ca2+ from pH-sensitive but thapsigargin-insensitive intracellular Ca2+ stores. Hypocapnic alkalosis caused a fivefold increase in [Ca2+]i compared with hypercapnic acidosis. Intracellular alkalinization at a normal extracellular pH did not affect [Ca2+]i. The hypocapnia-evoked increase in [Ca2+]i was decreased from 242 +/- 56 to 50 +/- 32 nmol/l by the removal of extracellular Ca2+. The main mechanism affecting the hypocapnia-dependent [Ca2+]i increase was thought to be the augmented influx of extracellular Ca2+ mediated by extracellular alkalosis. Hypercapnic acidosis caused little change in PGI2 production, but hypocapnic alkalosis increased it markedly. In conclusion, both hypercapnic acidosis and hypocapnic alkalosis increase [Ca2+]i in HPAECs, but the mechanisms and pathophysiological significance of these increases may differ qualitatively.

Roles of ICAM-1 for abnormal leukocyte recruitment in the microcirculation of bleomycin-induced fibrotic lung injury.

To assess the importance of endothelial intercellular adhesion molecule-1 (ICAM-1) in microvascular leukocyte kinetics in diseased lungs, we investigated the transitional changes in ICAM-1 expression, vascular diameter, and leukocyte behavior in rat pulmonary microcirculation during the development of acute lung injury (ALI) and chronic fibrosis (FIB) evoked by bleomycin (BLM). Observations were made in the isolated perfused lung with a real-time confocal laser luminescence microscope. Microvascular cell kinetics were evaluated by measuring the behavior of fluorescence- labeled leukocytes and erythrocytes in the presence or absence of anti-ICAM-1 monoclonal antibody (1A29). Arteriolar ICAM-1 showed little change at any time after BLM treatment. Venular ICAM-1 was first enhanced at the initial phase of ALI followed by the second upregulation at the early phase of FIB. Capillary ICAM-1 showed a sustained increase at both ALI and FIB. Arteriolar and venular diameters were not altered but capillary diameter decreased during ALI and early FIB stages. Although firm adherence of leukocytes to arteriolar and venular walls was not observed, rolling leukocytes were increased in venules both at the initial phase of ALI and at the early phase of FIB. The leukocyte rolling in venules correlated well with transitional changes in ICAM-1 and was inhibited by 1A29. Sustained entrapment of leukocytes in capillaries was attributed to changes in vascular diameter as well as augmented ICAM-1. In conclusion, ICAM-1 plays an important role in microvascular leukocyte recruitment in both ALI and FIB in the BLM-injured lung.

Effect of steroid on hyperoxia-induced ICAM-1 expression in pulmonary endothelial cells.

Intercellular adhesion molecule-1 (ICAM-1) of the vascular endothelium plays a key role in the development of pulmonary oxygen toxicity. We studied the effect of steroid on hyperoxia-induced ICAM-1 expression using cultured endothelial cells in vitro. Human pulmonary artery endothelial cells (HPAECs) were cultured to confluence, and then the monolayers were exposed to either control (21% O(2)-5% CO(2)) or hyperoxic (90% O(2)-5% CO(2)) conditions with and without a synthetic glucocorticoid, methylprednisolone (MP). MP reduced hyperoxia-induced ICAM-1 and ICAM-1 mRNA expression in a dose-dependent manner. Neutrophil adhesion to hyperoxia-exposed endothelial cells was also inhibited by MP treatment. In addition, MP attenuated hyperoxia-induced H(2)O(2) production in HPAECs as assessed by flow cytometry. An electrophoretic mobility shift assay demonstrated that hyperoxia activated nuclear factor-kappaB (NF-kappaB) but not activator protein-1 (AP-1) and that MP attenuated hyperoxia-induced NF-kappaB activation dose dependently. With Western immunoblot analysis, IkappaB-alpha expression was decreased by hyperoxia and increased by MP treatment. These results suggest that MP downregulates hyperoxia-induced ICAM-1 expression by inhibiting NF-kappaB activation via increased IkappaB-alpha expression.

Nitric oxide differentially attenuates microvessel response to hypoxia and hypercapnia in injured lungs.

The issue of whether the acinar microvessel response to alveolar hypoxia and hypercapnia is impaired in injured lungs has not been vigorously addressed, despite the importance of knowing whether it is or not when treating patients with serious lung injury in terms of permissive hypercapnia. Applying a real-time laser confocal luminescence microscope, we studied hypoxia- and hypercapnia-induced changes in the diameter of the intra-acinar arterioles, venules, and capillaries of isolated rat lungs harvested from animals exposed for 48 h to 21% O(2) (group N) or 90% O(2) (group H). Measurements were made with and without inhibition of nitric oxide (NO) synthase (NOS) by N(omega)-nitro-L-arginine methyl ester or of cyclooxygenase (COX) by indomethacin at different basal vascular tones evoked by thromboxane A(2) (TXA(2)) analog. Hypoxia in the absence of TXA(2) contracted arterioles in group N but not in group H. Attenuated hypoxia-induced arteriole constriction was restored almost fully by inhibiting NOS and partially by inhibiting COX. Hypercapnia induced venule dilation in group N, but did not dilate venules in group H, irrespective of TXA(2). NOS inhibition in hypercapnia unexpectedly enhanced venule and arteriole dilation in group H. These responses no longer occurred when NOS and COX were inhibited simultaneously. In conclusion, microvessel reactions to hypoxia and hypercapnia are abnormal in hyperoxia-injured acini, in which NO directly attenuates hypoxia-induced arteriole constriction, whereas COX inhibited by excessive NO impedes hypercapnia-induced microvessel dilation.

Plasma platelet-activating factor acetylhydrolase deficiency in Japanese patients with asthma.

Platelet-activating factor (PAF), a phospholipid with a wide range of proinflammatory actions, is immediately degraded and inactivated in vivo by PAF acetylhydrolase (PAF-AH). Surprisingly, 4% of the Japanese population lacks the extracellular isoform of this enzyme, plasma PAF-AH, due to a genetic missense (V279F) mutation. We studied the association of this mutation with asthma prevalence and phenotypes in the Japanese adult population. The allele frequency of V279F mutation was 18.6% in 279 patients with asthma (28.7% heterozygotes and 4.3% homozygotes) and 21.7% in 217 healthy subjects (32.3% heterozygotes and 5.5% homozygotes). V279F mutant allele prevalence was consistent regardless of asthma type (16.3% in atopic [n = 156] and 21.6% in nonatopic [n = 123]), or the severity of disease (21.7% in patients with mild [n = 97], 17.5% in those with moderate [n = 131], and 15.8% in those with severe [n = 51] asthma). Plasma PAF-AH activity was inversely proportional to the number of mutant alleles, and did not correlate with asthma prevalence, type, or severity. We concluded that plasma PAF-AH deficiency due to V279F mutation is not essential to the pathophysiology of asthma in the Japanese adult population.

Do adhesion molecules importantly regulate leukocyte kinetics within intraacinar microvessels of the lung?

Precise assessment of blood cell kinetics in the pulmonary microcirculation is extremely difficult because pulmonary microvascular architecture contains arterioles, venules and capillaries in an exceedingly intricate and densely convoluted fashion. Conventional epiluminescence microscopy may not be suitable for investigation of blood cell kinetic in the pulmonary microcirculation, in which arterioles, venules and capillary networks are not located in the same plane. To overcome these impediments, we recently developed a real-time confocal laser luminescence microscope with a high-speed analysis component having the capacity to yield confocal-images of rapidly moving cells at a rate of 1,000 frames/sec and at sufficiently high magnification. In the current review, we will first introduce the details of our newly developed observation system constructed with a view to estimation of blood cell dynamics in the intraacinar microcirculation of the lung. Applying this novel method to isolated perfused rat lungs, we will secondly address the issue of whether or not leukocyte-endothelium interactions in the pulmonary microcirculation qualitatively differ from those serving in the systemic microcirculation. We will particularly shed light on possible roles of endothelial ICAM-1, endothelial P-selectin and leukocyte L-selectin in distorting leukocyte kinetics in the intraacinar microvessels under a variety of diseased conditions, including prolonged exposure to a hyperoxic environment inducing a significant upregulation of ICAM-1 as well as P-selectin on the pulmonary microvascular endothelium, and stimulation of leukocytes by an IL-8 analog causing downregulation of leukocyte L-selectin but inverse upregulation of CD18-related integrins.

Impaired hypoxic vasoconstriction in intraacinar microvasculature in hyperoxia-exposed rat lungs.

To assess the effects of exposure of the lung to hyperoxic conditions on reactivity of pulmonary microcirculation to hypoxic stimulation, we measured hypoxia-elicited overall pulmonary pressor changes (HPV) and microvascular diameter changes in intraacinar arterioles, venules, and capillaries in isolated perfused rat lungs exposed to a hyperoxic environment (90% O2). To estimate the importance of vasoactive prostaglandins and nitric oxide (NO) for HPV modification, we examined the roles of constitutive and inducible forms of cyclooxygenase (COX-1 and COX-2) and those of NO synthase (eNOS and iNOS). Indomethacin was used for inhibiting both COX-1 and COX-2, while NS-398 was used as a selective inhibitor of COX-2. Both eNOS and iNOS were suppressed by L-NAME, whereas iNOS alone was inhibited by aminoguanidine. Microvascular diameter was measured with a real-time confocal laser scanning luminescence microscope. We found that (1) exposure to hyperoxia caused overall HPV and arteriolar constriction to be attenuated; (2) the blunted HPV was restored by L-NAME but not by aminoguanidine, indomethacin, or NS-398; and (3) arteriolar constriction was improved by either L-NAME, aminoguanidine, or indomethacin but only slightly by NS-398. In conclusion, attenuation of overall HPV in hyperoxia-exposed lungs is explicable mainly by excessive NO generated via eNOS, while impaired arteriolar constriction is caused by NO yielded by eNOS and iNOS as well as by vasodilating prostaglandin(s) produced by COX-1.

Response of intra-acinar pulmonary microvessels to hypoxia, hypercapnic acidosis, and isocapnic acidosis.

To elucidate the differential reactivity of pulmonary microvessels in the acini to hypoxia, excessive CO2, and increased H+, we investigated changes in the diameter of precapillary arterioles, postcapillary venules, and capillaries in isolated rat lungs on exposure to normocapnic hypoxia (2% O2), normoxic hypercapnia (15% CO2), and isocapnic acidosis (0.01 mol/L HCl). Microvascular diameters were precisely examined using a real-time confocal laser scanning luminescence microscope coupled to a high-sensitivity camera with an image intensifier. Measurements were made under conditions with and without indomethacin or N(omega)-nitro-L-arginine methyl ester to assess the importance of vasoactive substances produced by cyclooxygenase (COX) or NO synthase (NOS) as it relates to the reactivity of pulmonary microvessels to physiological stimuli. We found that acute hypoxia contracted precapillary arterioles that had diameters of 20 to 30 microm but did not constrict postcapillary venules of similar size. COX- and NOS-related vasoactive substances did not modulate hypoxia-elicited arteriolar constriction. Hypercapnia induced a distinct venular dilatation closely associated with vasodilators produced by COX but not by NOS. Arterioles were appreciably constricted in isocapnic acidosis when NOS, but not COX, was suppressed, whereas venules showed no constrictive response even when both enzymes were inhibited. Capillaries were neither constricted nor dilated under any experimental conditions. These findings suggest that reactivity to hypoxia, CO2, and H+ is not qualitatively similar among intra-acinar microvessels, in which COX- and NOS-associated vasoactive substances function differently.

Differential contribution of various adhesion molecules to leukocyte kinetics in pulmonary microvessels of hyperoxia-exposed rat lungs.

To elucidate the differential role of various adhesion molecules in distorting leukocyte behavior in the microvasculature of hyperoxia-exposed rat lungs, we investigated fluorescein-labeled leukocyte and erythrocyte kinetics in isolated lungs taken from the animals exposed to 90% O2 for 48 h under conditions in which endothelial intercellular adhesion molecule-1 (ICAM-1) and P-selectin were inhibited by appropriate monoclonal antibodies (1A29 for ICAM-1 and ARP2-4 for P-selectin), while leukocyte L-selectin was restrained with fucoidin. Measurements of blood cell kinetics were made by a confocal laser luminescence microscope coupled with a high-speed video camera. In addition, we histologically examined leukocyte accumulation within the alveolar septa and ICAM-1 as well as P-selectin expressions in the lung. We found that P-selectin expression was sparsely enhanced only in arterioles, whereas ICAM-1 was significantly induced in both venules and capillaries. Firm adhesion of leukocytes was not identified in arterioles and venules, whereas leukocyte rolling was evident in both the vessels. Arteriolar rolling was regulated via a P-selectin- and ICAM-1-independent but L-selectin-dependent mechanism, whereas venular rolling was mediated via a P-selectin-independent but ICAM-1- and L-selectin-dependent pathway. Leukocyte sequestration within capillaries was augmented by an ICAM-1-related mechanism. These findings may suggest that, in hyperoxia-exposed lungs, induction of adhesion molecules and their obstacles to leukocyte behavior are qualitatively different among arterioles, venules, and capillaries.

Effects of active vasoconstriction and total flow on perfusion distribution in the rabbit lung.

We analyzed the effects of hypoxic vasoconstriction and total flow on the distribution of pulmonary perfusion in 38 isolated left rabbit lungs perfused under zone 3 conditions. Lungs were suspended in an upright position, oriented to the apicobasal line. Distributions of regional perfusion rates (RPR) along the vertical and horizontal axes were determined using nonradioactive microspheres labeled with heavy metal elements, which were detectable with X-ray fluorescence spectrometry. Changing the O2 concentration of a respirator and an extracorporeal membrane oxygenator independently, respective influences of active vasoconstriction induced by alveolar hypoxia and pulmonary artery hypoxia (PA hypoxia) on the RPR distribution were examined at a flow rate of 0.4 ml x min(-1) x g wet lung tissue(-1). To analyze the effects of changes in total flow, we investigated the RPR distribution at a perfusion rate of 1.2 ml x min(-1) x g wet lung tissue(-1). The RPR distribution in the absence of hypoxia was inhomogeneous and was augmented in the lower lung fields, whereas alveolar hypoxia shifted the RPR upward and significantly diminished the RPR in the lung base. RPR distributions along the horizontal axes under alveolar hypoxia conditions demonstrated that remarkable hypoxic pulmonary vasoconstriction (HPV) takes place in medial regions at the lung base. PA hypoxia altered the RPR distribution in qualitatively the same manner as alveolar hypoxia. Increased flow rate augmented the RPR in the lung, except in the dorsobasal region. These results suggest that the occurrence of HPV and the vascular conductance are not uniform throughout the lung.

Leukocyte kinetics in the pulmonary microcirculation: observations using real-time confocal luminescence microscopy coupled with high-speed video analysis.

To quantitatively assess blood cell kinetics in the intact pulmonary microcirculation, in which arterioles, venules, and capillaries are exceedingly intricate and densely convoluted, we recently developed a real-time confocal laser luminescence microscope with a high-speed analysis component. The system has the capacity to yield confocal images of rapidly moving cells at a rate of 1000 frames/second and at sufficiently high degrees of magnification. Applying this novel method to isolated perfused rat lungs, we estimated the endothelial distributions of constitutively expressed intercellular adhesion molecule-1 (ICAM-1) and P-selectin and also studied leukocyte hemodynamic behavior in the pulmonary microvasculature under conditions in which ICAM-1, P-selectin, and L-selectin were inhibited, respectively, by 1A29 (monoclonal antibody to rat ICAM-1), ARP2-4 (monoclonal antibody to rat P-selectin), and fucoidin (competitive inhibitor of both P- and L-selectin). The results were compared with those obtained with a nonconfocal microscope using conventional epiluminescence. Intertwined microvessel networks in the lung were clearly distinguishable in confocal images but not in conventional nonconfocal views. ICAM-1 was perceptibly expressed along venular and capillary but not arteriolar endothelium, whereas P-selectin was undetectable in all microvessels examined. Leukocytes were not firmly adhered to venular or arteriolar endothelial cells. Leukocyte rolling was recognized more frequently along arteriolar walls than along venular walls and was suppressed in arterioles by L-selectin inhibition but not by either ICAM-1 or P-selectin inhibition. In capillaries, transient and sustained arrest of leukocytes occurred at physiologic shear rates. Inhibition of ICAM-1 or P-selectin had no remarkable effect upon either transient or sustained entrapment of leukocytes in capillaries. In conclusion, physiologic and biologic characteristics of pulmonary microvessels appear to be quite different from those of the systemic microcirculation.

[A case of interstitial pneumonia induced by intravesical administration of bacillus Calmette-Guerin (BCG)].

A 61-year-old man with superficial bladder cancer, which was detected after he complained of hematuria, was treated three times with intravesical BCG administration. Since liver dysfunction was detected thereafter, he was admitted to our hospital. Three days after admission, he complained of dyspnea on exertion associated with severe hypoxemia, as well as abnormal findings on chest X-ray, i.e. extensive bilateral lung densities. We performed bronchoscopic examination and obtained bronchoalveolar lavage fluid (BALF) and lung biopsy specimens (TBLB). In the BALF, a marked increase in the total cell number, particularly lymphocytes with a high CD4/CD8 ratio was noted. TBLB specimens revealed the lesions to be numerous non-caseating granulomas. We failed to obtain definite evidence of BCG in the sputum, urine, blood, and BALF. Instead, we found that a lymphocyte stimulation test for BCG (DLST) was strongly positive. Based on these findings, severe interstitial pneumonia probably induced by hypersensitivity against BCG, was diagnosed. Anti-tuberculous agents, and steroid-pulse therapy followed by oral administration of relatively low dose of steroid ameliorated the abnormal conditions, including chest X-ray film findings and hypoxemia. The population of lymphocytes and CD4/CD8 ratio in the BALF were reduced as well. Serious interstitial pneumonia was induced by the intravesical administration of BCG, which resulted in transitional changes in the BALF cell component.

[Two cases of pulmonary disease caused by Mycobacterium chelonae subsp. abscessus].

We encountered two-cases of pulmonary disease caused by M. chelonae subsp. abscessus, [Case 1] A 72-year-old man was admitted to the hospital because of fever. He had been observed for one year after being given a diagnosis of pulmonary disease caused by Myocobacterium avium complex. Sputum examination revealed acid-fast bacilli (Gaffky 9). He recovered after administration of clarithromycin (CAM) and other drugs. [Case 2] A 61-year-old man was admitted to the hospital because of coughing and sputum production. He had been observed for 4 years after being given a diagnosis of pulmonary M. fortuitum disease. Sputum examination revealed acid-fast bacilli (Gaffky 7). His symptoms deteriorated even though he received anti-tuberculosis agents and CAM. After measurement of minimal inhibitory concentration (MIC), he was given amikacin (AMK). In both cases, the bacilli found in sputum obtained on admission were identified as M. chelonae subsp. abscessus by DNA hybridization. They were completely resistant to all anti-tuberculosis agents. However, the disk method show that they were sensitive to AMK, imipenem and CAM. The MIC value of those strains to CAM was 0.78 microgram/ml in case I and more than 100 micrograms/ml in case 2. The results obtained by MIC measurement were consistent with the clinical outcome. AMK, cefoxitin (CFX), and CAM had been used to treat M. chelouae subsp. abscessus in Europe, but the MIC value differed from strain to strain within a species. Thus the present data suggest that measurement of the MIC value of CAM would be necessary to predict its therapeutic effect.

[A case of chronic necrotizing pulmonary aspergillosis treated with itraconazole].

An 84-year-old woman was admitted because of fever, hemoptysis and productive cough with infiltrative shadows in the right lung field on chest X-ray. Chronic necrotizing pulmonary aspergillosis was diagnosed on the basis of her clinical and radiographic features, positive cultures and positive serological tests. Conventional chemotherapy with fluconazole and 5-FC produced only minimal improvement. A course of itraconazole was initiated and proved to be effective.