Modification of the p53 transgene of a replication-competent adenovirus prevents mdm2- and E1b-55kD-mediated degradation of p53.

Clinical efficacy of adenovirus-mediated cancer gene therapy has been limited thus far. To improve its oncolytic effect, a replication-competent adenoviral vector was previously constructed to express high levels of p53 at a late time point in the viral life cycle. p53 expression from this vector improved tumor cell killing and viral spread in vitro. However, p53 function is antagonized by cellular mdm2 and adenoviral E1b-55kD, both of which are known to bind to and inactivate p53. Therefore, a new vector (Adp53W23S) that expresses a modified p53 transgene, which does not bind to E1b-55kd and mdm2, was constructed. The modified p53 protein was demonstrated to have a substantially prolonged half-life, and its localization was predominantly nuclear. Viral replication was unaffected by expression of the modified p53 and cancer cell killing was improved in vitro. However, in a xenograft model, efficacy was not significantly different from control virus. In conclusion, expression of a degradation-resistant p53 transgene late in the life cycle of a replication-competent adenovirus improves p53 stability and cancer cell killing in vitro. However, other factors, such as the adenoviral E1b-19kD and E1a proteins, which oppose p53 function, and limitations to viral spread need to be addressed to further improve in vivo efficacy.

Hypoxia reduces adenoviral replication in cancer cells by downregulation of viral protein expression.

Successful cancer therapy using replicating viral vectors relies on the spread of virus from infected to uninfected cells. To date, there has been limited clinical success in the use of replicating adenoviruses. In animal models, established xenograft tumors are rarely eliminated despite the persistence of high viral titers within the tumor. Hypoxia is a prevalent characteristic of solid tumors, whereas adenovirus naturally infects tissues exposed to ambient oxygen concentrations. Here, we report that hypoxia (1% oxygen) reduces adenoviral replication in H1299 and A549 lung cancer cells, BxPC-3 pancreatic cancer cells, LNCaP prostate cancer cells and HCT116 colon cancer cells. However, hypoxia does not reduce cell viability or restrict S-phase entry. Importantly, the production of E1a and fiber proteins under hypoxic conditions was substantially decreased at 24 and 48 h compared to room air controls. In contrast, Northern analysis showed similar levels of E1a mRNA in room air and hypoxic conditions. In conclusion, a level of hypoxia similar to that found within solid tumors reduces the replication of adenoviral vectors by reduction of viral protein expression without a reduction in mRNA levels. To further improve oncolytic therapy using a replicating adenovirus, it is important to understand the mechanism through which hypoxia and the virus interact to control expression of viral and cellular proteins, and consequently to develop means to overcome decreased viral production in hypoxic conditions.

Wild-type adenovirus decreases tumor xenograft growth, but despite viral persistence complete tumor responses are rarely achieved--deletion of the viral E1b-19-kD gene increases the viral oncolytic effect.

Strategies to target viral replication to tumor cells hold great promise for the treatment of cancer, but even with replicating adenoviruses complete tumor responses are rarely achieved. To evaluate replicating adenoviral vectors, we have used A549 human lung cancer nude mouse xenografts as a model system. Intratumoral injection of wild-type adenovirus (Ad309) significantly reduced tumor growth from day 14 (p = 0.04) onward; however, tumor volumes reached a plateau at day 50. At 100 days, high levels of titratable virus were present within persistent viable tumors. In contrast to viral injection into established tumors, when tumor cells were infected in vitro with wild-type virus and then mixed with uninfected tumor cells, 1% of infected cells was sufficient to prevent tumor establishment. An E1b-19kD-deleted viral mutant (Ad337) was more efficient than Ad309 in this cell-mixing model. Just 1 cell in 1000 infected with Ad337 prevented tumor growth. However, although better than wild-type virus, Ad337 was unable to eradicate established flank tumors. These data suggest that although replicating adenoviruses exhibit significant oncolytic activity, barriers within the established tumor, such as connective tissue and tumor matrix, may limit the spread of virus. Strategies to enhance viral spread through established tumors are therefore likely to greatly improve the therapeutic efficacy of replicating adenoviruses.

Hut lung. A domestically acquired particulate lung disease.

We report an illustrative case of advanced "hut lung," or domestically acquired particulate lung disease (DAPLD), in a recently emigrated nonsmoking Bangladeshi woman with a history of 171 hour-years of exposure to biomass smoke. She presented with symptoms of chronic cough, dyspnea, and early parenchymal lung disease. High-resolution computed tomography (CT) of the chest demonstrated numerous 2- to 3-mm nodules, sparing the pleural surface. To our knowledge, this is the first such report of CT findings in the literature. Bronchoscopy yielded typical anthracotic plaques and diffuse anthracosis with interstitial inflammation on histopathologic examination of biopsy specimens. DAPLD is potentially the largest environmentally attributable disorder in the world, with an estimated 3 billion people at risk. Caused by the inhalation of particles liberated from the combustion of biomass fuel, DAPLD results in significant morbidity from infancy to adulthood. Clinically, DAPLD manifests a broad range of disorders from chronic bronchitis and dyspnea to advanced interstitial lung disease and malignancy. While a detailed environmental history is essential for making the diagnosis in most individuals, for patients with advanced DAPLD, invasive modalities such as bronchoscopy with transbronchial biopsy and examination of bronchoalveolar lavage fluid help differentiate it from other diseases. Recognition of this syndrome and removal of the patient from the environment is the only treatment. The development of well-controlled interventional trials and the commitment of sufficient resources to educate local populaces and develop alternative fuel sources, stove designs, and ventilation are essential toward reducing the magnitude of DAPLD.

Deletion of the adenoviral E1b-19kD gene enhances tumor cell killing of a replicating adenoviral vector.

Replicating adenoviral vectors are a promising new modality for cancer treatment and clinical trials with such vectors are ongoing. Targeting these vectors to cancer cells has been the focus of research. However, even if perfect targeting were to be achieved, a vector still must effectively kill cancer cells and spread throughout the bulk of the tumor. The adenoviral E1b-19kD protein is a potent inhibitor of apoptosis and may therefore compromise the therapeutic efficacy of an adenoviral vector. In this study we have investigated if an E1b-19kD gene deletion could improve the ability of a replicating adenoviral vector to spread through and kill cancer cells. In several lung cancer cell lines an E1b-19kD-deleted virus (Ad337) induced substantially more apoptosis than did a wild-type virus (Ad309), and tumor cell survival was significantly reduced in three of four cell lines. In addition, the apoptotic effects of cisplatin or paclitaxel were augmented by Ad337, but inhibited by wild-type virus. The number of infectious virus particles in the supernatant of infected cells was increased with Ad337 compared with wild-type virus, indicating enhanced early viral release. Ad337, in contrast to Ad309, induced significantly larger plaques after infection of A549 cells. This well-described large plaque phenotype of an E1b-19kD mutant virus is likely the result of early viral release and enhanced cell-to-cell viral spread. Loss of E1b-19kD function caused only minor cell line-specific increase or decrease in viral yield. We conclude that deletion of the E1b-19kD gene may enhance the tumoricidal effects of a replicating adenoviral vector.

Shoulder impingement in front-crawl swimming: I. A method to identify impingement.

PURPOSE: The impingement of subacromial structures has been proposed as a major cause of the shoulder problems experienced by athletes who use repetitive overhead actions. The purpose of this study was to develop a noninvasive method to identify instances at which the shoulder was experiencing impingement during front-crawl swimming. METHODS: Shoulder impingement has been reported to occur when an arm is: (a) elevated above shoulder height while being rotated internally; and (b) forcibly elevated at, or beyond, the maximum active elevation angle. In this study shoulder configurations that satisfied the above two conditions were sought throughout the functional range of each shoulder; and a boundary that distinguished configurations that would cause shoulder impingement was defined. The shoulder movements exhibited during performance of the front-crawl stroke were measured using three-dimensional videography and compared with the boundary defined for each shoulder. The shoulder was considered to experience impingement if the shoulder configuration observed exceeded the boundary defined for that shoulder. RESULTS: For a male collegiate swimmer, impingement occurred for 12% of the stroke time for each shoulder. CONCLUSIONS: The analysis permitted the identification of the instances at which the shoulders were experiencing impingement during the front-crawl swimming. In this study, the measurement of the boundary was based entirely upon the mechanism of impingement described in the literature. Further studies are needed to confirm the occurrence of impingement by means of advanced visualization techniques, such as magnetic resonance imaging (MRI) and ultrasonogram.

Shoulder impingement in front-crawl swimming: II. Analysis of stroking technique.

PURPOSE: The purpose of this study was to determine the technical causes of shoulder impingement experienced by front-crawl swimmers. METHODS: The shoulder movements exhibited during performance of the front-crawl stroke were measured using three-dimensional videography, and the instances at which each shoulder was experiencing impingement were identified. RESULTS: On average, impingement occurred 24.8% of the stroke time (%ST). In one or more phases of the stroke cycle, each subject experienced impingement in some trials and not in other trials. This suggests that stroke technique, and not just anatomical differences, accounted for individual susceptibility to shoulder impingement. No significant difference was found between the mean values for %ST for slow and fast stroking speeds and for trials with and without hand paddles. Use of a unilateral breathing technique was often associated with a small magnitude of tilt angle (an effect of the scapular elevation/abduction on one side and depression/adduction on the other side) on the breathing side; in such cases a high incidence of shoulder impingement was observed for the shoulder on the ipsilateral side. Swimmers at high risk of experiencing shoulder impingement had three characteristics in their stroking techniques: (a) a large amount of internal rotation of the arm during the pull phase, (b) a late initiation of external rotation of the arm during the recovery phase, and (c) a small amount of tilt angle. CONCLUSIONS: A swimmer should be able to reduce the risk of developing shoulder impingement by altering the technique to eliminate the three characteristics.

Active responses decrease impact forces at the hip and shoulder in falls to the side.

Active responses, such as using the arm to break the fall, may be an effective means of decreasing likelihood of injury in a fall and may help explain why only a small percentage of falls result in a fracture. We quantified the impact force at the hip and shoulder in falls to the side from a kneeling position under three conditions: (1) attempting to break the fall by using an arm; (2) falling with the body relaxed; and (3) falling with the body tensed. Subjects fell from a kneeling position onto a force platform array covered with foam padding and impact force data were recorded. The ground reaction force-time curve was generally bimodal due to sequential impacts of the hip and shoulder. Impact forces at the hip and shoulder were 12 and 16% less for the slap condition (p < 0.05) than for the tensed condition. The impact forces for the relaxed and tensed conditions were not significantly different, although impact forces tended to be less in the relaxed condition. We concluded that active responses reduce the impact forces experienced at the hip and shoulder in falls to the side. Decreased effectiveness of protective responses, due to increases in reaction time and decreases in strength with age, may help explain why so many hip fractures occur in the elderly but so few occur in younger people.

Targeting the replication of adenoviral gene therapy vectors to lung cancer cells: the importance of the adenoviral E1b-55kD gene.

It has been proposed that an adenovirus with the E1b-55kD gene deleted has a selective advantage in replicating in cancer cells that have mutations in the p53 gene (Bischoff et al., 1996). We have explored this hypothesis in several lung cancer cell lines, and evaluated potential mechanisms that might regulate the replication of Ad338, an E1b-55kD-deleted virus, with the objective of developing a rational approach for targeting gene therapy to lung tumors. Our data show that Ad338 replicates poorly in three lung cancer cell lines with various p53 mutations (H441, H446, and Calu1), yet this virus replicates to a high level in a lung cancer cell line with wild-type p53 (A549) and in a normal lung fibroblast line (IMR90). Viral DNA replication, expression of viral proteins, and shutoff of host cell proteins were not important variables in limiting the replication of the E1b-55kD-deleted virus. However, the cell lines resistant to host cell protein shutoff were also the most resistant to the cytopathic effect induced by mutant and wild-type virus and the only cells to survive for 8 days following infection. The E1b-55kD protein clearly has an important role in viral replication beyond its interaction with p53. Thus, an E1b-55kD-deleted virus cannot be used to specifically target viral replication to p53-mutated lung cancer cells.

Changes in muscle-tendon length during the take-off of a running long jump.

The aims of this study were to determine: (1) the modes of action of selected muscles of the supporting leg during the take-off of a running long jump; (2) whether the instant at which maximum knee flexion is recorded is a valid indicator of the instant these muscles change their modes of action; and (3) the relationships between the actions of these muscles and the change in the vertical velocity of the centre of mass during the take-off. Eleven elite, female long jumpers performed six jumps from a full-length approach. A model of selected muscles of the jumping leg was developed to estimate muscle-tendon lengths from segment positions obtained using cinematography. Only half of the muscles exhibited a lengthening-shortening sequence of activity. The instant at which maximum knee flexion was recorded was a poor indicator of when the muscles changed from eccentric to concentric activity. The more the vasti muscles were stretched, the larger the gain in vertical velocity; the longer the triceps surae muscles at touchdown, the more they were stretched, and the faster they were stretched, the larger the gain in vertical velocity. Enhancement through use of the stretch-shortening cycle did not make a significant contribution to vertical velocity via the actions of the vasti and triceps surae muscles.

Mechanical characteristics of knee extension exercises performed on an isokinetic dynamometer.

The purpose of this study was to evaluate selected mechanical characteristics of knee extension exercises performed on a LIDO Active Isokinetic System. A female subject performed two repetitions of maximal effort knee extension at 16 different preset angular velocities (PAVs). The gravitational and inertial effects were included in the computation of the resultant knee torque. For each repetition, the knee flexion angle, the angular velocity and acceleration of the shank, and the knee torque throughout the range of motion were computed. The shank angular acceleration values indicated that if the inertial effect is not considered the knee torque will be underestimated in the initial phase and errors in knee torque up to about 6 N.m can be expected for the rest of the repetition. The durations when the shank angular velocity was within +/- 5% and +/- 10% of PAV (expressed as percentages of the repetition time) were found to decrease with increasing PAV. The difference between PAV and shank angular velocity at the instant of peak torque also increased with increasing PAV. The results demonstrate the limitations that may exist in an isokinetic dynamometers.

Optimum phase ratio in the triple jump.

The purpose of this study was to develop and validate a method to determine the optimum phase ratio that yields the longest actual distance for a given triple jumper. Two hypotheses were tested: (a) for any given triple jumper, the greater the gain in the vertical velocity the greater the loss in the horizontal velocity; and (b) there is no single optimum phase ratio for all triple jumpers. Kinematic data were collected for four elite male triple jumpers. It was found that the loss in the horizontal velocity during a support phase has a significant positive linear correlation with the gain in the vertical velocity during the same support phase. The slope of the regression line was referred to as the horizontal-to-vertical velocity conversion factor. Based on this relationship, an optimization model for the longest actual distance was developed to determine the optimum phase ratio for each of the four subjects. The optimization results showed that there was an optimum phase ratio for the longest actual distance for each triple jumper. The results of a validity test showed that the model predicted the actual distance with a degree of fair accuracy. The results of sensitivity analysis showed that the optimum phase ratio for a given athlete was a function of the horizontal-to-vertical velocity conversion factor and the horizontal velocity at the touchdown of the hop. These results support the two hypotheses of this study.

Three-dimensional videography of swimming with panning periscopes.

The purpose of this paper is to describe a three-dimensional videography method with panning periscopes for reconstructing swimming techniques. Two panning periscope systems, developed in our laboratory, were used for the data collection. A control object (1 x 1.2 x 2)m3 with 72 markers was placed in seven consecutive locations along the (1.5 x 8.4 x 2) m3 calibration space. The position of the control object was recorded while panning the periscope systems, and for each of the seven locations the DLT camera parameters were determined for eight fields equally spaced through the panning motion. Each DLT camera parameter was then expressed as a function of the camera orientation by using a least-squares, second-order, polynomial regression equation, so that the DLT camera parameters for any camera orientation could be predicted (Yu et al., J. Biomechanics 26, 741-751, 1993). The three-dimensional coordinates of desired body landmarks at an instant were determined from the digitized two-dimensional coordinates for two cameras and the predicted DLT camera parameters at that instant. The accuracy of the method was evaluated by (a) the resultant errors in computing three-dimensional coordinates of precalibrated static points, and (b) the errors in computing the length of a scale rod pulled through the calibration space. The mean resultant errors ranged from 8.34 to 16.44 mm for the above- and from 9.93 to 16.22 mm for the below-water control volumes. The mean error in computing lengths on the scale rod ranged from 3.32 to 5.83 mm for three 0.5 m lengths, and 9.97 mm for a 1.5 m length. The method produced acceptable results in the reconstruction of three-dimensional motions recorded from a large space above and below the water surface.

Modification of nasal epithelial potential differences of individuals with cystic fibrosis consequent to local administration of a normal CFTR cDNA adenovirus gene transfer vector.

Mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) manifest on the nasal epithelial surface of individuals with cystic fibrosis (CF) by Na+ hyperabsorption and diminished beta-agonist-induced Cl- conductance, resulting in an abnormal bioelectric phenotype across the nasal epithelium. A clinical trial was conducted to determine if a replication-deficient, recombinant adenovirus vector containing a normal copy of the CFTR cDNA (AdCFTR) could, when administered to the nasal epithelium, correct the abnormal bioelectric phenotype. Nine individuals with CF received 2 x 10(5) to 2 x 10(8.5) plaque forming units of AdCFTR to the epithelium of one nostril. Measurements made included: baseline electrical potential difference (PD) between the surface of the nasal epithelium and the interstitial fluid, change in PD in response to amiloride, which inhibits apical Na+ channels, and change in PD in response to isoproterenol in a low Cl- solution, a measure of cAMP-regulated Cl- conductance. The functional integrity of the epithelium was evaluated by the PD response to ATP. Each individual served as their own control with measurements made in the nostril to be treated before AdCFTR administration, and in the contralateral untreated nostril. On the average, in the treated nostril over 2 weeks after the local administration of the adenovirus vector compared to measurements made in the same nostril before treatment, baseline PD decreased toward normal (-53.3 +/- 4.0 to -34.6 +/- 3.4, p = 0.01), response to amiloride decreased toward normal (36.9 +/- 4.7 to 19.7 +/- 3.0, p = 0.02), and response to low Cl- and isoproterenol increased toward normal (-4.5 +/- 1.5 to -9.1 +/- 2.1, p = 0.05). There were no changes in response to ATP (-15.3 +/- 2.7 to -15.8 +/- 1.9, p = 0.39), suggesting that the epithelium remained functionally intact. Importantly, there were no significant changes in measurements made in the untreated nostril. While limited to the nasal epithelium, these data suggest an adenovirus vector can safely deliver sufficient CFTR cDNA function to improve the abnormal CF bioelectric phenotype.

Human CC10 gene expression in airway epithelium and subchromosomal locus suggest linkage to airway disease.

The CC10 gene encodes the Clara cell 10-kDa protein, which is expressed in airway epithelial cells. Quantification of CC10 gene expression in freshly isolated human proximal airway epithelial cells demonstrated very high mRNA levels, approximately fivefold greater than gamma-actin mRNA, and in situ hybridization localized CC10 mRNA to nonciliated airway epithelial cells. Sequence analysis demonstrated that the human CC10 gene is comprised of three short exons separated by a long first and short second intron, and with a 5' flanking region typical of a regulated gene. Three Alu repeats were observed in intron 1 and one in intron 2. Two polymorphic regions within the introns were identified. First, a microsatellite was localized 5' to the third Alu repeat in intron 1 with a variable number of 4- and 5-base pair (bp) repeats and a heterozygosity of 0.71. Second, in 3% of the 168 chromosomes examined, there was the insertion of a human-specific Alu repeat in intron 2, 45 bp 3' to the exon 2-intron junction. In three Centre d'Etude du Polymorphisme Humain families, meiotic breakpoint analysis using these two polymorphic loci localized the CC10 gene to 11 p12-q13 between markers D11S16 and D11S97, a region recently linked to atopy and to the beta-subunit of the high-affinity immunoglobulin E receptor. The observations in the present study of high-level expression of the CC10 gene in the epithelium of conducting airways and a subchromosomal localization of the gene to a region potentially linked to inflammatory airway disease, together with the reported anti-inflammatory and immune-modulating properties of the protein, suggest the CC10 gene product may be important in modulating inflammation within the airways. If so, the highly heterozygous microsatellite described in the present study should facilitate analysis of a possible linkage of the CC10 gene with an inherited susceptibility to asthma.

Critical characteristics of technique in throwing the discus.

The purpose of this study was to identify those characteristics of the techniques used by elite discus throwers that are most closely related to the distances they record. The subjects were the competitors in the discus throw events at two major meetings. Two S-VHS video cameras were used to record the performances of the subjects; the direct linear transformation (DLT) procedure was used to obtain three-dimensional data from these records. For the males, the change in speed of the discus during the second double support phase was more influential than the change in speed during any other phase in accounting for differences in the distances of the throws recorded. For the females, the changes in speed during the flight phase and during the second double support phase were about equally influential in accounting for differences in the distance thrown. The findings suggest that emphasis placed on achieving a large change in the speed of the discus during the second double support phase is well founded; that the speed of release is the most influential determinant of the distance of the throw; and that, in most cases, the aerodynamic forces exerted on the discus during the flight increase the distance of the throw.

Identification of a 27 bp 5'-flanking region element responsible for the low level constitutive expression of the human cytosolic phospholipase A2 gene.

The cytosolic phospholipase A2 (cPLA2) gene codes for an enzyme that liberates arachidonic acid from membrane phospholipids, and thus plays a pivotal role in the production of the prostaglandin and leukotriene mediators of inflammation, as well as in a variety of cell signalling pathways. After preliminary studies demonstrated the cPLA2 gene is expressed in a variety of human tissues and was localized to the q arm of chromosome 1 between markers F13B and D1S74, we cloned and characterized the 5'-flanking region of this gene in order to identify the elements controlling its low level constitutive expression. The 5'-flanking region has features typical of a housekeeping gene with no TATA box or CAAT box, although atypical in that it is not GC rich, has no SP1 sites, and has a long run of CA repeats. Analysis of fragments of the 5'-flanking region demonstrated that 541 bp 5' to exon 1 supported reporter gene activity at a level 30% of the SV40 promoter. Interestingly, similar activity was observed by deleting most of the 5'-flanking region down to a 27 bp region containing a sequence with homology to the initiator sequence in the terminal deoxynucleotidyl transferase gene and a polypyrimidine tract similar to the initiator element of the mouse ribosomal protein gene. Within this 27 bp region, a 10 bp fragment (-17 to -8 bp) within the polypyrimidine tract is critical for the baseline expression of the human cPLA2 gene. While the 5'-flanking region contains a putative composite AP-1 and glucocorticoid response element, this region does not respond to tumor necrosis factor-alpha (TNF) and/or glucocorticoids in a cell line (HEp-2) that exhibits upregulation of cPLA2 mRNA transcript levels by TNF. The observations that the expression of the cPLA2 gene is tightly controlled at a relatively low level is consistent with the evolving concept that modulation of expression of this critical enzyme is primarily at the post-translational level.

Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis.

We have administered a recombinant adenovirus vector (AdCFTR) containing the normal human CFTR cDNA to the nasal and bronchial epithelium of four individuals with cystic fibrosis (CF). We show that this vector can express the CFTR cDNA in the CF respiratory epithelium in vivo. With doses up to 2 x 10(9) pfu, there was no recombination/complementation or shedding of the vector or rise of neutralizing antibody titres. At 2 x 10(9) pfu, a transient systemic and pulmonary syndrome was observed, possibly mediated by interleukin-6. Follow-up at 6-12 months demonstrated no long term adverse effects. Thus, it is feasible to use an adenovirus vector to transfer and express the CFTR cDNA in the respiratory epithelium of individuals with CF. Correction of the CF phenotype of the airway epithelium might be achieved with this strategy.

Modulation of secretory leukoprotease inhibitor gene expression in human bronchial epithelial cells by phorbol ester.

Secretory leukoprotease inhibitor (SLPI), a 12-kD nonglycosylated serine antiprotease, helps to protect the epithelial surface of the airways from the destructive capacity of neutrophil elastase. Based on the recognition that SLPI levels can increase in the presence of airway inflammation, we hypothesized that inflammatory stimuli should modulate the expression of the SLPI gene in airway epithelial cells. To evaluate this, the modulation of SLPI gene expression with various inflammatory stimuli was evaluated in the HS-24 human bronchial epithelial cell line. After preliminary studies showed that several inflammatory mediators enhanced SLPI messenger RNA (mRNA) levels, PMA was used as a model inflammatory stimulus. PMA significantly increased the level of 0.7-kb SLPI mRNA transcripts in HS-24 cells in a dose- and time-dependent fashion and increased the amount of SLPI protein in the culture supernatant. Nuclear run-on analyses showed that the SLPI gene transcription rate increased approximately twofold after PMA stimulation. Transfection studies using fusion genes composed of fragments of up to 1.2 kb of the 5' flanking sequence of the SLPI gene and a luciferase reporter gene demonstrated potent promoter activity in the 131-bp segment (-115 to +16 relative to the transcription start site), and all longer segments up to 1.2 kb, whereas smaller segments showed low promoter activity. An 18-bp element (-98 to -115), in a region with homology to PMA-responsive regions in the Moloney murine leukemia virus enhancer and the IL-8 gene, was shown to be of importance in the level of transcription of the SLPI gene. However, this element was not responsible for the upregulation of SLPI gene expression by PMA. Evaluation of HS-24 cells in the presence of actinomycin D demonstrated that SLPI mRNA transcripts were very stable and became more so in the presence of PMA. Thus, SLPI gene expression in airway epithelial cells can be upregulated by an inflammatory stimulus, and this modulation is regulated at both the transcriptional and posttranscriptional levels. These mechanisms of SLPI upregulation likely play a role in defending the epithelial surface in the local milieu of inflammatory lung diseases.