Signature current of SO2-induced bronchitis in rabbit.

To investigate abnormalities of airway epithelial ion transport underlying chronic inflammatory airway diseases, we performed electrophysiological, histological, and molecular biological experiments using rabbits exposed to SO2 as a model of bronchitis. By comparison with control, the SO2-exposed trachea exhibited decreased short circuit current (Isc) and conductance associated with increased potential difference. In normal trachea, apical ATP induced a transient Isc activation followed by a suppression, whereas the bronchitis model exhibited a prolonged activation without suppression. This pathological ATP response was abolished by diphenylamine 2-carboxylate or Cl--free bath solution. A significant increase in net Cl- flux toward the lumen was observed after ATP in our bronchitis model. Isoproterenol or adenosine evoked a sustained Isc increase in SO2-exposed, but not in normal, tracheas. The Northern blot analysis showed a strong expression of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in SO2-exposed epithelium. The immunohistochemical study revealed a positive label of CFTR on cells located luminally only in SO2-exposed rabbits. We concluded that the prolonged ATP response in our bronchitis model was of a superimposed normal and adenosine-activated current. The latter current was also activated by isoproterenol and appeared as a signature current for the bronchitis model airway. This was likely mediated by CFTR expressed in the course of chronic inflammation.

The mouse L-histidine decarboxylase gene: structure and transcriptional regulation by CpG methylation in the promoter region.

To investigate the regulation of mouse L-histidine decarboxylase (HDC) gene expression, we isolated genomic DNA clones encoding HDC. Structural analysis revealed that the mouse HDC gene was composed of 12 exons, spanning approximately 24 kb. Northern blotting analysis indicated that, among the cell lines examined, a high level of HDC gene expression was restricted to mature mast cell lines and an erythroblastic cell line. The gene was induced strongly in the mouse immature mast cell line P815 after incubation in the peritoneal cavity of BDF1 mice. We observed that the promoter region was demethylated in the HDC-expressing cell lines and in induced P815 cells. Interestingly, forced demethylation by 5-azacytidine (5-azaC) treatment induced high expression of HDC mRNA in P815 cells. The activity of a mouse HDC promoter-reporter construct stably transfected in P815 cells was repressed by in vitro patch-methylation. This low promoter activity of the patch-methylated reporter construct was restored after 5-azaC treatment, which demethylated the patch-methylated promoter. These results indicate that DNA methylation state of the promoter region controls HDC gene expression.

Reduced infiltration of tumor-associated macrophages in human prostate cancer: association with cancer progression.

Tumor-associated macrophages (TAMs) are highly active immune effector cells that may either positively or negatively regulate the growth of various malignant cells, depending on the biological context. However, the role of TAMs in human prostate cancer progression is unclear. TAMs were immunohistochemically labeled using a monoclonal (CD68) antibody in radical prostatectomy specimens derived from 81 prostate cancer patients. CD68-positive cells were counted with the aid of a microscope and expressed as macrophage index (MphiI), including TAMs/mm2 total tumor tissue (MphiItotal), TAMs/mm2 tumor stroma (MphiIstroma), and TAMs/mm2 cancer cell area (MphiIcancer). MphiIs were analyzed in association with patients' clinical and pathological stage, recurrence status, and histological grade of the cancer. There were significant inverse relationships between MphiItotal and MphiIstroma and clinical stage (P = 0.016 and P = 0.006, respectively). Reduced MphiItotal was also associated with the presence of positive lymph nodes (P = 0.010). Interestingly, although all of the MphiIs differed between Gleason score groups, only MphiIcancer was positively associated with Gleason score. Univariate analysis of MphiItotal and multivariate analysis of MphiItotal with specific pathological markers revealed that MphiItotal was an independent predictor for disease-free survival after surgery (Cox proportional hazard model, P = 0.044 and P = 0.007, respectively). For patients with high MphiItotal (> or = 185.8, the mean MphiItotal value), the disease-free probability 5 years after surgery was 0.75, which was significantly higher than for those with low MphiItotal (0.31, P = 0.0008). Additional immunohistochemical studies that evaluated cytotoxicity-related biomarkers in stroma-associated mononuclear cells suggested reduced functional activities in highly aggressive prostate cancer compared with less aggressive disease. Our results indicate that reduced MphiItotal is a novel prognostic marker for prostate cancer.

Signal transduction of mucous secretion by bronchial gland cells.

Bronchial glands, which consist of mucous and serous cells, are abundant in human airways, playing a major role in the airway secretion. Cl(-) secretion is accompanied by water transport to the lumen in the acinar cells of bronchial glands. Agonists that increase [Ca(2+)]i induce the Cl(-) secretion in bronchial glands. Ca(2+) release from a IP(3)-sensitive Ca(2+) pool at the apical portion stimulates and opens Ca(2+)-sensitive Cl(-) channels at the apical membrane, producing Cl(-) secretion in bronchial glands. K(+) channels at the basolateral membranes are Ca(2+)-sensitive and activated by Ca(2+) release from a cADPribose-sensitive Ca(2+) pool, maintaining the Cl(-) secretion in bronchial glands. Further, cADP ribose in concert with IP(3) induce [Ca(2+)]i oscillation, inducing Cl(-) secretion in bronchial glands. Some tyrosine kinases are involved in the Cl(-) secretion in bronchial glands. Mucous and serous cells in bronchial glands take part in mucin secretion and the secretion of defensive substances (glycoconjugates), respectively. [Ca(2+)]i oscillations are shown to play a central role in the exocytosis of secretory granules in serous cells of bronchial glands. Other signal transductions of mucin and glycoconjugates in airway gland cells remain to be studied, although agonists which increase [cAMP]i are also well known to induce mucin and glycoconjugate secretion from airway glands.

In situ prostate cancer gene therapy using a novel adenoviral vector regulated by the caveolin-1 promoter.

Caveolin-1, a structural component of caveolae, is overexpressed in metastatic and androgen-resistant prostate cancer and highly expressed in tumor-associated endothelial cells. The mouse cav-1 promoter was cloned and placed upstream of the HSV-tk gene in an adenoviral vector (Adcav-1tk) and compared with a cytomegalovirus (CMV) or Rous sarcoma virus (RSV) promoter-driven HSV-tk, AdCMVtk and AdRSVtk vectors, respectively. Mouse and human prostate cancer cells and mouse endothelial cells were infected with Adcav-1tk, AdCMVtk or control vectors without the HSV-tk gene (Adcav-1 and AdCMV) and subsequently treated with ganciclovir (GCV). GCV-mediated in vitro cytotoxicity induced by the Adcav-1tk vector was comparable to that for AdCMVtk in multiple mouse and human prostate cancer cell lines. To evaluate the activity of Adcav-1tk in vivo, orthotopic mouse prostate cancer tumors were generated with RM-9 cells and injected in situ with Adcav-1tk, AdCMVtk, AdRSVtk, or AdCMVbetagal (control) and treated with GCV. All three HSV-tk transducing vectors produced statistically significant reductions in wet weight and increased apoptotic indices compared with the control vector. However, only Adcav-1tk produced significant necrosis, and only Adcav-1tk and AdRSVtk caused significant decreases in microvessel density. In conclusion, Adcav-1tk demonstrated efficacy in vitro and in vivo in preclinical models of prostate cancer. Our results suggest that the cav-1 promoter may have unique benefits in targeting gene therapy to prostate cancer and its associated vasculature.

Prostate cancer gene therapy: comparison of adenovirus-mediated expression of interleukin 12 with interleukin 12 plus B7-1 for in situ gene therapy and gene-modified, cell-based vaccines.

We have documented previously that adenovirus-mediated interleukin 12 (IL-12) gene therapy is effective for orthotopic tumor control and suppression of pre-established metastases in a preclinical prostate cancer model (Y. Nasu et al., Gene Ther., 6: 338-349, 1999). In this report, we directly compare the effectiveness of an adenovirus that expresses both IL-12 and the costimulatory molecule B7-1 (AdmIL12/B7) with one that expresses IL-12 alone (AdmIL-12) using the poorly immunogenic RM-9 orthotopic murine model of prostate cancer. We document AdmIL-12/B7-mediated secretion of IL-12 and increased surface expression of B7-1 in infected RM-9 tumor cells. A significant reduction in orthotopic tumor size and increased survival was demonstrated in mice treated with a single orthotopic injection of AdmIL-12/B7 compared with AdmIL-12 or controls. Six of 19 animals treated with AdmIL-12/B7 survived long term with apparent eradication of the primary tumor in contrast to one of 38 animals in the AdmIL-12-treated group. Orthotopic treatment of tumors with both vectors led to an infiltration of both CD4+ and CD8+ immunoreactive cells, with AdmIL-12/B7 treatment having a more prolonged infiltration of CD8+ cells. AdmIL-12/B7 was also more effective than AdmIL-12 or controls at suppression of pre-established metastases. We further developed a vaccine model based on s.c. injection of infected, irradiated RM-9 cells and found that both AdmIL-12 and AdmIL-12/B7 are effective at suppressing the development and growth of challenge orthotopic tumors using this protocol.

Structural evidence of genomic exon-deletion mediated by Alu-Alu recombination in a human case with heme oxygenase-1 deficiency.

We previously reported a family affected by heme oxygenase-1 (HO-1) deficiency [Yachie et al., 1999]. The proband was a compound heterozygote for a complete loss of exon 2 (the maternal allele) and a two-nucleotide deletion within exon 3 (the paternal allele). In this report, we describe a large genomic deletion (1730 bp) including entire exon 2 in this family as a specific mechanism generating exon-2 absence observed in the HO-1 mRNA. Analysis of the deletion junction demonstrated fusion of a 5' portion of Alu-Sx element with a 3' portion of Alu-Sq element. The junction contained sequences with high homology to the recombinogenic Alu "core" sequence. These structural features of the HO-1 gene suggest homologous recombination associated with Alu element. This study presents the initial characterization of the HO-1 gene defect causing a human case of HO-1 deficiency and provides the molecular basis for understanding this genetic disease.

Detection of surfactant protein-A gene transcript in the cells from pleural effusion for the diagnosis of lung adenocarcinoma.

PURPOSE: To determine whether detecting surfactant protein-A (SP-A) gene transcript in the cells from pleural effusion is useful for the diagnosis of lung adenocarcinoma. PATIENTS AND METHODS: We performed reverse transcription polymerase chain reaction (RT-PCR) analysis of SP-A gene transcript in the cells of pleural effusion from 42 consecutive patients with pleural effusion, including 7 patients with primary lung adenocarcinoma before their treatments. RESULTS: A cDNA segment of SP-A was amplified from the pleural fluid cells of all patients with primary lung adenocarcinoma, indicating the presence of the SP-A gene transcript. None of the remaining patients, including those with metastatic lung adenocarcinoma, showed positive for the SP-A gene transcript. CONCLUSION: These findings indicate that RT-PCR analysis of the SP-A gene transcript in pleural effusion is useful for the diagnosis of primary lung adenocarcinoma.

Bronchorrhea from diffuse lymphangitic metastasis of colon carcinoma to the lung.

Bronchorrhea, defined as watery sputum of 100 ml or more per day, was seen in a 52-year-old female patient with diffuse lymphangitic metastasis of colon carcinoma to the lung. For 5 months before the visit to our clinic, she complained of progressive worsening of the cough, watery sputum, and shortness of breath. On admission to our hospital, she expectorated large amounts of nonpurulent watery sputum (150 to 300 ml/d), and showed diffuse reticular and linear shadows in both lungs on chest radiograph and severe obstructive impairment (FEV1 percent, 35 percent) in lung function tests. Histologic findings obtained from both surgical specimens at abdominal operation for ileus and lungs at the autopsy revealed lymphangitic metastasis of ascending colon carcinoma to the lung. At autopsy, histologically the lungs showed diffuse infiltrations of mucus-secreting adenocarcinoma cells to both lung parenchyma and airway submucosa.

Intrapleural omentum simulating pleural effusion.

We report herein a case of Morgagni hernia of omentum into the pleural space, simulating a pleural effusion on a routine chest radiograph. A 62-year-old man was referred to our clinic for close examination of a pleural effusion-like shadow at the right costophrenic region. He had no history of trauma and no symptoms. Chest computed tomographic scan showed a pleural effusion-like shadow with a fat density. Thoracoscopy revealed a movable omentum-like mass and no significant fluid in the right pleural space. Magnetic resonance imaging and celiac angiography confirmed the herniation of omentum into the right pleural space. This case suggests that a Morgagni hernia must be excluded in a patient with a fat density effusion-like shadow in the pleural space.

Chemical properties of bronchorrhea sputum in bronchial asthma.

Bronchorrhea, defined as watery sputum of 100 ml or more per day, was seen in 18 of 207 patients (8.7 percent) with bronchial asthma during attack. Fifteen bronchorrhea sputum samples were chemically examined using ten parameters: dry weight, albumin, IgA, pH, Na+, Cl-, K+, prostaglandins E and F and histamine, and compared with eight saliva samples and 17 mucoid sputum samples. Bronchorrhea sputum differed from saliva in its chemical parameters. Bronchorrhea sputum exhibited parameter values intermediate between those of saliva and mucoid sputum, except for the two following parameters. The pH of bronchorrhea sputum was significantly lower than that of mucoid sputum and histamine concentration, expressed as weight per dry weight of sample, was significantly higher in bronchorrhea than in mucoid sputum. Administration of corticosteroid or an histamine H1-blocker to five to nine asthmatic patients with associated bronchorrhea sputum during asthmatic attacks, significantly reduced the volume of bronchorrhea sputum, whereas anticholinergics and H2-blocker did not alter the sputum volume.

Role of chronic Pseudomonas aeruginosa infection in airway mucosal permeability.

The role of Pseudomonas aeruginosa infection in airway mucosal permeability was studied in 16 patients with chronic bronchitis by measuring the amounts of radiolabeled albumin in sputum. One group (A) consisted of six patients (two female, four male, 53 +/- 6 years, SEM) with chronic P aeruginosa infection for 5 +/- 0.9 years. Another group (B) consisted of ten patients (five female, five male, 67 +/- 4 years) without P aeruginosa infection for at least two years. No significant differences between groups A and B were found in the volume of sputum (63 +/- 21 ml/day in group A and 45 +/- 8 ml/day in group B, p = 0.44), the obstructive changes (FEV1 of 57 +/- 6 percent in group A and 51 +/- 4 percent in group B), or the duration of disease (19 +/- 4 years in group A and 14 +/- 4 years in group B). Saliva, sputum, and serum samples were collected at intervals of 2 h over an 8-h period, and at 24 h after intravenous administration of 131I-labeled human albumin. For counting, free 131I was removed by dialysis. Radiocounts (cpm) of saliva were significantly smaller than those of sputum or serum. The cpm from each sputum sample was divided by serum cpm at the time of each sampling. Group A showed significantly higher values in the ratio of sputum- to serum-cpm than did group B at all sampling times. Furthermore, the ratios at 2 and 4 h after 131I-albumin injection significantly correlated with sputum volume per day, whereas they did not correlate with any other factors (age, obstructive impairment, and duration of disease). These findings suggest that chronic P aeruginosa infection produces an increase in airway mucosal permeability to albumin.

Perivascular fibrosis of muscular pulmonary arteries in chronic obstructive pulmonary disease.

We performed a morphometric analysis of peribronchiolar and perivascular fibrosis in lungs obtained at autopsy from six patients with chronic bronchitis, six with pulmonary emphysema, and four normal control subjects. The areas of fibrosis outside the smooth muscle layer of bronchioles and outside the external elastic lamina of muscular pulmonary arteries were measured and their thickness was then calculated by assuming a round airway or artery. Patients with chronic bronchitis had significantly thicker peribronchiolar fibrosis in bronchioles of 1 mm or less in diameter and also thicker perivascular fibrosis of the adjacent muscular pulmonary arteries than the other two groups. The extent of perivascular fibrosis was significantly correlated with peribronchiolar fibrosis only in the muscular pulmonary arteries adjacent to the bronchioles but not in those away from the bronchioles. These findings suggest direct extension of chronic inflammation from bronchioles to the adjacent muscular pulmonary arteries in chronic bronchitis but not in pulmonary emphysema. Such perivascular fibrosis might lead to sustained pulmonary hypertension.

Marked goblet cell hyperplasia with mucus accumulation in the airways of patients who died of severe acute asthma attack.

To examine the changes in airways in bronchial asthma (BA) during an asthma attack causing death, we performed morphometric analysis of autopsied lungs from three outpatients who died of severe acute asthma attacks (group A) and compared these to five patients who died of non-status asthmaticus (group B). Controls (group NL) were four patients who died of diseases other than respiratory disorders. Area proportions of bronchial glands to bronchial wall (gland [percent]) and of goblet cells to total epithelial layer (goblet [percent]) and the intraluminal amount of mucus in the airways (MOR) were measured in a paraffin section. There were no significant differences in age, sex, smoking history, duration of BA history, and dosage of glucocorticoids received between groups A and B. Although both groups A and B showed significantly larger values of gland (percent) in the central airways and of inflammatory cell numbers in the airway walls than did group NL, no significant differences were observed between groups A and B. In contrast, markedly significant increases in goblet (percent) and in MOR were observed in group A compared to groups B and NL. These increases in group A were more dominant in the peripheral airway: 30-fold and threefold increases of group B in goblet (percent) and MOR, respectively. Furthermore, MOR significantly correlated with goblet (percent) in the peripheral airways (p less than 0.05). These findings suggest that a marked increase in goblet cells of the airways is a feature characteristic of patients with BA who die of a severe acute attack.

Role of chronic Pseudomonas aeruginosa infection in the development of bronchiectasis.

To understand the role of Pseudomonas aeruginosa infection in the development of bronchiectasis, we investigated by CT the presence of bronchiectasis in two groups of chronic bronchitis patients and in a control group. There were no differences in clinical or laboratory findings between groups A and B. Three observers without any knowledge of these patients reported bronchiectasis on a scale of 0 to 3 and bronchial wall thickness on a scale of 0 to 3 in each lobe of both lungs. Bronchiectasis and wall thickness scores in group A (chronic bronchitis with P aeruginosa infection) were significantly higher than bronchiectasis scores and wall thickness in group B (chronic bronchitis without P aeruginosa infection). Both scores in group B were higher than those in group C (control group). These findings support the idea that chronic P aeruginosa infection plays a role in the development of bronchiectasis.

Contractility of isolated single submucosal gland from trachea.

We isolated single submucosal glands from canine and feline trachea. Examination by light and electron microscope showed that these isolated glands consist mainly of glandular tissue, and no smooth muscle. Cell components in the glandular tissue were ultrastructurally normal, and myoepithelial cells surrounded acini and secretory tubules. In response to methacholine, the mucus was squeezed from the tip of the collecting ducts in coincidence with the contraction of the glands. The contractile properties of isolated single glands were examined with a force transducer. Cholinergic agents (methacholine and acetylcholine) as well as 40-150 mM K+ showed a dose-response relationship and induced tension up to 12 mg. The length-tension relationship was also observed. The removal of Ca2+ from the medium eliminated contractile response. Caffeine induced approximately 30% of the response to methacholine, and phenylephrine, a tension less than 30% of that with methacholine. These findings suggest that squeezing of mucus due to the contraction of myoepithelial cells has an important effect on secretory response of airway submucosal glands.

Effect of epithelium on mucus secretion from feline tracheal submucosal glands.

We studied the effect of airway epithelium on mucus secretion by use of an isolated tracheal submucosal gland preparation reported previously (J. Appl. Physiol. 60: 1237-1247, 1986). Mucus glycoconjugate release from submucosal glands of feline trachea was examined using [3H]glucosamine as a mucus precursor. Isolated glands showed significantly higher secretory responses to cholinergic, alpha-, and beta-adrenergic agonists and dibutyryladenosine 3',5'-cyclic monophosphate (average 400% of control) than the conventional tracheal mucosal explants, which contained epithelium and submucosal tissues in addition to submucosal glands (average 160% of control). The addition of isolated epithelium depressed the secretory response of isolated glands to the same level as that of tracheal explants. However, the supernatant from isolated epithelium failed to inhibit secretory responses to methacholine in isolated glands, suggesting that the epithelium-derived inhibitory factor to secretion may be short-lived. Leukotriene D4 antagonist (FPL 55712), cyclooxygenase and/or lipoxygenase inhibitors (indomethacin or BW 755C) caused no significant change in the inhibitory action of epithelium, suggesting that the inhibition is not due to arachidonic acid metabolites. The newly found secretory inhibitory action of epithelium is of particular interest in the pathogenesis of hypersecretion associated with epithelial damage.

Neural control of contraction in isolated submucosal gland from feline trachea.

To determine the autonomic innervation to myoepithelial cells of submucosal gland, we applied electrical field stimulation (FS) to the intrinsic nerves in isolated submucosal glands from feline tracheae. FS induced contraction that was voltage or frequency dependent and abolished by pretreatment with tetrodotoxin. DMPP (1,1-dimethyl-4-phenylpiperazinium iodide) did not produce any significant contraction, and pretreatment with hexamethonium did not alter the response to FS. Atropine inhibited the contractile response to FS and neostigmine augmented the response to FS. Serotonin also augmented the response to FS, whereas the response to methacholine remained unchanged in the presence of serotonin. Phentolamine reduced the response to FS by 15% of control, whereas propranolol induced no significant changes in the response to FS. No significant inhibitory responses were observed by FS. Our findings indicate that the contraction of tracheal submucosal glands is mediated mainly by cholinergic nerves via muscarinic receptors and in small part by adrenergic nerves via alpha-receptors, and serotonin potentiates the contractile response to FS at the postganglionic nerve.

VIP augments cholinergic-induced glycoconjugate secretion in tracheal submucosal glands.

Using isolated submucosal glands from feline trachea, we examined the effect of vasoactive intestinal peptide (VIP) on mucus glycoprotein secretion and glandular contraction by measuring released radiolabeled glycoconjugates and induced tension, respectively. VIP (10(-10) to 10(-6) M) produced a dose-dependent increase in [3H]glycoconjugate release of up to 300% of controls, which was inhibited by VIP antiserum and not inhibited by atropine, propranolol, or phentolamine. VIP at a low concentration (10(-9) M), which did not produce any significant increases over controls, produced a 2.4- to 5-fold augmentation of the glycoconjugate release induced by 10(-9) to 10(-7) M methacholine (MCh). Atropine or VIP antiserum abolished the augmentation. VIP did not produce any alteration in isoproterenol- or phenylephrine-evoked glycoconjugate secretion. VIP (up to 10(-5) M) did not produce any alteration in the tension, even when the gland had contracted with MCh, or any augmentation of contraction induced by MCh (10(-9) to 10(-7) M). These results indicate that VIP induces mucus glycoprotein release from secretory cells and also that it potentiates the secretion induced by cholinergic stimulation.

Effect of substance P on mucus secretion of isolated submucosal gland from feline trachea.

To elucidate how substance P (SP) produces submucosal gland secretion, we examined the effects of SP on the glandular contractile response and 3H-labeled glycoconjugate release in isolated submucosal glands from feline tracheae. SP (10(-12) to 10(-4) M) produced dose-dependent increases in the contractile response, and the maximal tension induced by SP was approximately 70% of the response to methacholine. SP-induced contraction is blocked completely by atropine and augmented by neostigmine. Pretreatment with hemicholinium 3, an acetylcholine synthesis inhibitor, inhibited the contractile response to SP. Pretreatment with tetrodotoxin did not inhibit the contractile response to SP. Capsaicin induced tension of a magnitude similar to that of SP. SP (10(-7) M) produced a significant increase (74% above control) in radiolabeled glycoconjugate release from isolated glands, whereas SP had no significant effects on glycoconjugate release from tracheal explants, probably because of epithelial suppression. Atropine abolished SP-evoked glycoconjugate release in isolated glands. Our findings indicate that 1) SP induces glandular contraction, which is related to the squeezing of mucus in the ducts and secretory tubules, 2) SP stimulates radiolabeled glycoconjugate release in isolated submucosal gland, probably involving mucus synthesis and/or cellular secretion, and 3) these two actions are mediated by a peripheral cholinergic mechanism.