Description of a Unique, Complex Feeding Socket Caused by the Putative Primitive Root-Knot Nematode, Meloidogyne kikuyensis.

Meloidogyne kikuyensis produces unique galls that form on one side of the root resembling nitrogen-fixing nodules that are produced on legumes in response to infection by Rhizobium and related bacteria. The gall caused by this root-knot nematode is made up of a complex feeding socket composed of several giant cells that are ramified with xylem vessels extending perpendicular from the vascular cylinder. The anterior portion of the second-stage juvenile, which develops into an adult, plugs into this unique feeding socket. The socket and the surrounding parenchyma together form a gall that is very different in morphology from those typically caused by other species of root-knot nematodes. Even though M. kikuyensis was considered to be a primitive species because of its low chromosome count, the complexity of its feeding site and minor plant damage suggests a more derived systematic position.

Does blood of healthy subjects contain bacterial ribosomal DNA?

Real-time PCR methods with primers and a probe targeting conserved regions of the bacterial 16S ribosomal DNA (rDNA) revealed a larger amount of rDNA in blood specimens from healthy individuals than in matched reagent controls. However, the origins and identities of these blood-associated bacterial rDNA sequences remain obscure.

Screening-level ecological risk assessment of polychlorinated dibenzo-p-dioxins and dibenzofurans in sediments and aquatic biota from the Venice Lagoon, Italy.

Recent monitoring data indicate that portions of Italy's Venice Lagoon ecosystem have been degraded due to biological and chemical pollution from a variety of potential sources. Using polychlorinated dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) data collected from sediment, fish and shellfish in the Lagoon, a screening-level ecological risk assessment (ERA) was performed to evaluate the risks to representative aquatic biota and wildlife receptors. Risks to aquatic invertebrates posed by PCDD/Fs in sediment were evaluated by comparing measured tissue concentrations in fish and shellfish to appropriate ecotoxicological reference values. For mammalian and avian receptors, risks posed by theoretical exposures to PCDD/Fs through the food chain were calculated using conservative wildlife exposure models. Results of the screening-level approach indicate that the potential for adverse effects to fish and aquatic invertebrate receptors from PCDD/Fs in surficial sediments are unlikely. Adverse effects to wildlife are possible but highly uncertain, and warrants further investigation in a more comprehensive ERA.

Evaluation of protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains.

Three hundred and twenty isolates of Staphylococcus aureus were typed by DNA sequence analysis of the X region of the protein A gene (spa). spa typing was compared to both phenotypic and molecular techniques for the ability to differentiate and categorize S. aureus strains into groups that correlate with epidemiological information. Two previously characterized study populations were examined. A collection of 59 isolates (F. C. Tenover, R. Arbeit, G. Archer, J. Biddle, S. Byrne, R. Goering, G. Hancock, G. A. Hébert, B. Hill, R. Hollis, W. R. Jarvis, B. Kreiswirth, W. Eisner, J. Maslow, L. K. McDougal, J. M. Miller, M. Mulligan, and M. A. Pfaller, J. Clin. Microbiol. 32:407-415, 1994) from the Centers for Disease Control and Prevention (CDC) was used to test for the ability to discriminate outbreak from epidemiologically unrelated strains. A separate collection of 261 isolates form a multicenter study (R. B. Roberts, A. de Lencastre, W. Eisner, E. P. Severina, B. Shopsin, B. N. Kreiswirth, and A. Tomasz, J. Infect. Dis. 178:164-171, 1998) of methicillin-resistant S. aureus in New York City (NYC) was used to compare the ability of spa typing to group strains along clonal lines to that of the combination of pulsed-field gel electrophoresis and Southern hybridization. In the 320 isolates studied, spa typing identified 24 distinct repeat types and 33 different strain types. spa typing distinguished 27 of 29 related strains and did not provide a unique fingerprint for 4 unrelated strains from the four outbreaks of the CDC collection. In the NYC collection, spa typing provided a clonal assignment for 185 of 195 strains within the five major groups previously described. spa sequencing appears to be a highly effective rapid typing tool for S. aureus that, despite some expense of specificity, has significant advantages in terms of speed, ease of use, ease of interpretation, and standardization among laboratories.

The use of benchmark dose methodology with acute inhalation lethality data.

Benchmark dose methodology has been proposed as a refinement to the no observed adverse effect level (NOAEL) methods currently used for health risk assessments. We compared log-normal probit and quantal Weibull benchmark concentration (BMC) estimates using 1, 5, and 10% response incidences with inhalation toxicity NOAELs and LOAELs from 120 acute lethality data sets. These studies yielded relatively steep dose-response slopes, which in turn influenced the suitability of selecting response incidences. The mean magnitude of difference between the 95% lower confidence limits (LCLs) for 1, 5, or 10% BMCs and corresponding NOAELs was less than twofold using the probit model and less than fourfold using the Weibull model. BMC estimates at the 10% response exceeded the observed LOAEL in some cases. Maximum likelihood estimates for doses with 1, 5, or 10% responses frequently exceeded LOAELs. The probit model repeatedly gave a better fit for the data compared with the Weibull model, resulting in improved goodness of fit tests and reduced 95% confidence intervals. The 95% LCL appears to be necessary at the 1, 5, or 10% response levels in order to safely estimate a concentration below that resulting in a LOAEL.

Entomoplasma freundtii sp. nov., a new species from a green tiger beetle (Coleoptera: Cicindelidae).

A mollicute (strain BARC 318T) isolated from gut tissue of a green tiger beetle (Coleoptera: Cicindelidae) was found by dark-field microscopy to consist of non-helical, non-motile, pleomorphic coccoid forms of various sizes. In ultrastructural studies, individual cells varied in diameter from 300 to 1200 nm, were surrounded by a cytoplasmic membrane and showed no evidence of cell wall. The organisms were readily filterable through membrane filters with mean pore diameters of 450 and 300 nm, with unusually large numbers of organisms filterable through 200 nm pore membrane filters. Growth occurred over a temperature range of 15-32 degrees C with optimum growth at 30 degrees C. The organism fermented glucose and hydrolysed arginine but did not hydrolyse urea. Strain BARC 318T was insensitive to 500 U penicillin ml-1 and required serum or cholesterol for growth. It was serologically distinct from all currently described sterol-requiring, fermentative Mycoplasma species and from 12 non-sterol-requiring Mesoplasma species, 13 non-sterol-requiring Acholeplasma species and 5 previously described sterol-requiring Entomoplasma species. Strain BARC 318T was shown to have a G + C content of 34 mol% and a genome size of 870 kbp. The 16S rDNA sequence of strain BARC 318T was compared to 16S rDNA sequences of several other Entomoplasma species and to other representative species of the genera Spiroplasma and Mycoplasma, and to other members of the class Mollicutes. These comparisons indicated that strain BARC 318T had close phylogenetic relationships to other Entomoplasma species. On the basis of these findings and other similarities in morphology, growth and temperature requirements and genomic features, the organism was assigned to the genus Entomoplasma. Strain BARC 318T (ATCC 51999T) is designated the type strain of Entomoplasma freundtii sp. nov.

Comparison of phenotypic and genotypic techniques for identification of unusual aerobic pathogenic gram-negative bacilli.

Rapid and accurate identification of bacterial pathogens is a fundamental goal of clinical microbiology, but one that is difficult or impossible for many slow-growing and fastidious organisms. We used identification systems based on cellular fatty acid profiles (Sherlock; MIDI, Inc., Newark, Del.), carbon source utilization (Microlog; Biolog, Inc., Hayward, Calif.), and 16S rRNA gene sequence (MicroSeq; Perkin-Elmer Applied Biosystems Division, Foster City, Calif.) to evaluate 72 unusual aerobic gram-negative bacilli isolated from clinical specimens at the Mayo Clinic. Compared to lengthy conventional methods, Sherlock, Microlog, and MicroSeq were able to identify 56 of 72 (77.8%), 63 of 72 (87.5%), and 70 of 72 (97.2%) isolates to the genus level (P = 0.002) and 44 to 65 (67.7%), 55 of 65 (84.6%), and 58 of 65 (89.2%) isolates to the species level (P = 0.005), respectively. Four Acinetobacter and three Bordetella isolates which could not be identified to the species level by conventional methods were identified by MicroSeq. In comparison to the full 16S rDNA sequences, the first 527 bp provided identical genus information for all 72 isolates and identical species information for 67 (93.1%) isolates. These data show that MicroSeq provides rapid, unambiguous identification of clinical bacterial isolates. The improved turnaround time provided by genotypic identification systems may translate into improved clinical outcomes.

Staphylococcus succinus sp. nov., isolated from Dominican amber.

Two bacterial isolates, designated AMG-D1T and AMG-D2, were recovered from 25-35-million-year-old Dominican amber. AMG-D1T and AMG-D2 biochemically most closely resemble Staphylococcus xylosus; they differ physiologically from other staphylococci. Fatty acid analysis and comparisons with extensive databases were unable to show relatedness to any specific taxon. Moreover, AMG-D1T and AMG-D2 contain tuberculostearic acid and meso-diaminopimelic acid, characteristic of the G + C-rich coryneform bacteria, as opposed to L-lysine characteristic of staphylococci. AMG-D1T and AMG-D2 have a G + C ratio of 35 mol%. Phylogenetic analysis with the 16S rRNA gene indicated that AMG-D1T and AMG-D2 were most closely related to Staphylococcus equorum, S. xylosus, Staphylococcus saprophyticus and other novobiocin-resistant staphylococci. Stringent DNA-DNA hybridization studies with AMG-D1T revealed similarities of 38% with S. equorum, 23% with S. xylosus and 6% with S. saprophyticus. The results indicate that AMG-D1T and AMG-D2 represent a novel species, which was named Staphylococcus succinus sp. nov. The type strain of the new species is AMG-D1 (ATCC 700337).

In vitro reduction kinetics of hexavalent chromium in human blood.

This study examines time- and concentration-dependent changes in distribution of hexavalent chromium [Cr(VI)] and total chromium [Cr-(TOT)] in reconstituted human blood following addition of potassium dichromate. Fresh human blood stabilized with EDTA was obtained from human volunteers soon after meal ingestion and at 2.5 h after a light meal (herein defined as "2.5-h fasted" conditions). Cr(VI) spiked into plasma under 2.5-h fasting conditions at 3.0-12.5 micrograms/L was stable for several hours, indicating a lack of appreciable reductive capacity in isolated plasma. Spiked plasma following a recent meal exhibited immediate but variable reduction of Cr(VI) up to 300 micrograms/L. When the spiked plasma was recombined with the red blood cell (RBC) fraction, rapid reduction occurred in both the plasma and the RBC fractions based on measurement of Cr(VI) and Cr(TOT). The data indicate that plasma reduction capacity is enhanced by a recent meal, but may be overwhelmed at Cr(VI) concentrations between 2000 and 10,000 micrograms/L. These data also suggest that the RBC fraction apparently has the capacity to reduce Cr(VI) at concentrations in blood up to 15,000 micrograms/L, and that the rate of Cr(VI) uptake into RBCs may not exceed the rate of intracellular reduction at these concentrations.

Rapid detection of Mycoplasma pneumoniae by an assay based on PCR and probe hybridization in a nonradioactive microwell plate format.

A new molecular assay, based on a rapid DNA extraction protocol, PCR, and hybridization to a specific probe in a nonradioactive microwell plate format was used to detect Mycoplasma pneumoniae in bronchoalveolar fluid specimens. The sensitivity of the assay was determined to be 10 to 100 organisms with M. pneumoniae reference strains. Specificity testing with different bacteria capable of producing pneumonia showed no cross-reactivity. In a prospective study, bronchoalveolar lavage fluids obtained from patients with pneumonia were investigated with the PCR assay and compared to culture. Twelve positive samples were detected with the PCR assay. Seven of them were subsequently confirmed by culture. All patients with positive PCR results seroconverted. Application of the PCR assay described may lead to safe and early diagnosis of M. pneumoniae in patients with pneumonia.

Ingestion of chromium(VI) in drinking water by human volunteers: absorption, distribution, and excretion of single and repeated doses.

This study examines the magnitude of hexavalent chromium [Cr(VI)] absorption, distribution, and excretion following oral exposure to 5 and 10 mg Cr(VI)/L in drinking water administered as a single bolus dose (0.5 L swallowed in 2 min) or for 3 d at a dosage of 1 L/d (3 doses of 0.33 L each day, at 6-h intervals). Adult male volunteers ingested deionized water containing various concentrations of potassium chromate, and samples of urine, plasma, and red blood cells (RBCs) were collected and analyzed for total chromium throughout the studies. In the bolus dose studies, a fairly consistent pattern of urinary chromium excretion was observed, with an average half life of about 39 h. However, 4-d total urinary chromium excretion and peak concentrations in urine and blood varied considerably among the 5 volunteers. Studies of repeated exposure to smaller volumes ingested at a more gradual rate (i.e., 0.33 L over 5-15 min) showed similar urinary chromium excretion patterns but generally lower chromium uptake/excretion. Given that sustained elevations in RBC chromium levels provide a specific indication of chromium absorption in the hexavalent state, these data suggest that virtually all (> 99.7%) of the ingested Cr(VI) at 5 and 10 mg Cr(VI)/L was reduced to Cr(III) before entering the blood-stream. The interindividual differences in total chromium uptake and excretion are plausibly explained by ingestion of appreciable doses on an empty stomach, which likely results in the formation of well-absorbed Cr(III) organic complexes in gastrointestinal tissues and possibly the blood. The lack of any clinical indications of toxicity in the volunteers and the patterns of blood uptake and urinary excretion of chromium are consistent with a predominant uptake of Cr(III) organic complexes [derived from Cr(VI)] that are excreted more slowly than inorganic forms of Cr(III). Therefore, it appears that the endogenous reducing agents within the upper gastrointestinal tract and the blood provide sufficient reducing potential to prevent any substantial systemic uptake of Cr(VI) following drinking-water exposures at 5-10 mg Cr(VI)/L. Based on these data, the chemical environment in the gastrointestinal tract and the blood is effective even under relative fasting conditions in reducing Cr(VI) to one or more forms of Cr(III).

Observation of steady state in blood and urine following human ingestion of hexavalent chromium in drinking water.

The uptake and elimination of Cr(VI) in a male volunteer who ingested 2 L/d of water containing 2 mg/L for 17 consecutive days was measured. Total chromium was measured in urine, plasma, and red blood cells (RBCs) for 4 d prior to and 2 wk after dosing (34 d total). The estimated bioavailability (2%) and the plasma elimination half-life (36 h) were consistent with our previous studies of Cr(VI) ingestion in humans. Steady-state chromium concentrations in urine and blood were achieved after 7 d of Cr(VI) ingestion. Both plasma and red blood cell (RBC) chromium concentrations returned rapidly to background levels within a few days after cessation of dosing. Since the concentration of chromium in the RBC should not decrease quickly if the chromium had entered the RBC as Cr(VI), these data support our prior work suggesting that concentrations of 10 mg Cr(VI)/L or less in drinking water of exposed humans appears to be completely reduced to Cr(III) prior to systemic distribution. Clinical chemistry data indicate that no toxicity occurred.

Refined exposure assessment for ingestion of tapwater contaminated with hexavalent chromium: consideration of exogenous and endogenous reducing agents.

Laboratory studies were conducted to determine how rapidly and completely chromium (VI) [Cr(VI)] is reduced upon contact with common beverages mixed with tapwater. Studies were performed for five common beverages (coffee, tea, orange juice, Kool Aid, and powdered lemonade) spiked with either 10 or 50 mg Cr(VI)/l. The concentrations of Cr(VI) were measured at several time intervals for up to four hours. It was demonstrated that each of these beverages had the capacity to reduce a concentration of > or = 8 mg Cr(VI)/l within a 15-minute time frame, and that continued monitoring of the beverages revealed greater reduction of the Cr(VI). These findings are consistent with the observation that many foods and beverages, as well as endogenous body fluids such as saliva and gastric juices, are capable of reducing substantial quantities of Cr(VI) to Cr(III). Our exposure assessment shows that the estimated high-end ingested dose of Cr(VI) from tapwater at both 1 and 5 mg Cr(VI)/l is generally two to three orders of magnitude below doses shown to have no adverse health effect in animal studies. When considered in conjunction with studies demonstrating that the reductive capacity of gastric juices may exceed 50 mg Cr(VI) daily, these observations suggest that little or no Cr(VI) is likely to be absorbed orally at a reasonable water concentration of Cr(VI), since tapwater is bright yellow at 5 mg Cr(VI)/l.

Assessment of airborne hexavalent chromium in the home following use of contaminated tapwater.

Field studies were conducted to estimate the plausible uptake of hexavalent chromium [Cr(VI)] aerosols inhaled during indoor residential use of a shower or an evaporative cooler supplied with water containing Cr(VI). In the evaporative cooler study, water concentrations of 20 mg Cr(VI)/L did not produce an increased concentration of airborne Cr(VI). The indoor air concentration of Cr(VI), measured over 24 hours of use, was 0.3-2.7 ng/m3, about the same as the concurrent outdoor concentrations. In the shower study, the average airborne concentrations of Cr(VI) aerosols at breathing-zone height ranged from 87 to 324 ng Cr(VI)/m3 when the water concentration of Cr(VI) was 0.89 to 11.5 mg/L. The Cr(VI) concentration in air was correlated directly to water concentration. The lifetime average daily doses and incremental cancer risk estimates corresponding to 30-year residential exposures were calculated using the measurements in this study and published exposure guidelines. The plausible upperbound lifetime cancer risk associated with continuous exposure to "background" Cr(VI) in outdoor air was estimated at 6.9 per million for a person exposed during ages 0-30, and 4.0 per million for ages 30-60. Similarly estimated upperbound cancer risks due to inhalation of shower aerosols from water containing 2-10 mg Cr(VI)/L over the same exposure period ranged from 0.9 to 5.5 per million. Our calculations demonstrate that shower aerosols do not contribute appreciably to background Cr(VI) exposures and risks, even at concentrations exceeding 2 mg Cr(VI)/L, which exhibit a discernible and unaesthetic yellow color that may limit the potential for long-term exposures of this type. We conclude that exposure to indoor aerosols from water containing Cr(VI) is unlikely to create a health hazard at concentrations up to 10 mg Cr(VI)/L. Furthermore, these aerosol measurements may be relevant to estimating airborne exposures to other nonvolatile chemicals.

Dose-dependent tolerance to ozone. III. Elevation of intracellular Clara cell 10-kDa protein in central acini of rats exposed for 20 months.

Understanding factors that promote pulmonary tolerance to long-term oxidant injury is essential to evaluating health risks in humans. One such factor may be Clara cell 10-kDa protein (CC10), a protein secreted by nonciliated cells in distal conducting airways thought to have an anti-inflammatory action against inhaled xenobiotics. Using standard immunohistochemical techniques and laser scanning confocal microscopy, we assessed changes in CC10 abundance in the centriacinar region of the rat following 20 months' exposure to 0.12 and 1.00 ppm ozone. Three zones of reflectance intensity (high, medium, and low), directly related to CC10 density, were used to distinguish between the two major subcellular compartments where CC10 is distributed: granules and endoplasmic reticulum. Low levels of ozone (0.12 ppm) had no significant effect on the cellular distribution or abundance of CC10 in nonciliated epithelium in the centriacinar region. In contrast, 1.00 ppm ozone not only elevated cellular volume of granule-based CC10, but also elevated the protein's concentration within the granules and increased the number of granules per cell profile. The proportion of nonciliated cells in terminal bronchioles increased significantly at the expense of ciliated cells. This combination of factors led to a threefold increase in CC10 stored per unit surface area of epithelium in terminal bronchioles. The nonciliated cells in ozone-induced respiratory bronchioles contained a distribution of CC10 similar to that of bronchiolar nonciliated cells in control animals. We conclude that ozone-induced tolerance may be related to the increased abundance and wider distribution of CC10 in central acini of rats following long-term ozone exposure.

Evaluation of central nervous system involvement in Lyme borreliosis patients with a solitary erythema migrans lesion.

To determine whether early dissemination of Borrelia burgdorferi to the central nervous system occurs in stage I of Lyme borreliosis, neurological and cerebrospinal fluid examination was performed in 48 consecutive patients in whom the only sign of infection was a solitary erythema migrans lesion. Long-term follow-up after treatment with tetracycline was carried out by telephone interview. At presentation, neurological findings were normal in all 48 patients. Cerebrospinal fluid samples were obtained from 29 (60%) patients. Mild pleocytosis and mild impairment of the blood-brain barrier were present in four and one of these patients, respectively. No significant amount of tumor necrosis factor or interleukin 6 was found in the cerebrospinal fluid samples. Culture results of 13 cerebrospinal fluid samples were negative. Borrelia burgdorferi DNA was only detected by the polymerase chain reaction in one of two aliquots of the cerebrospinal fluid sample of one patient. None of 46 patients who were interviewed 12 to 51 (median 25) months after antibiotic treatment developed manifestations consistent with disseminated or chronic Lyme borreliosis. Thus, no compelling evidence was found for the presence of asymptomatic central nervous system involvement in patients with clinically localized Lyme borreliosis.

Regulation of Clara cell 10 kD protein secretion by pilocarpine: quantitative comparison of nonciliated cells in rat bronchi and bronchioles based on laser scanning confocal microscopy.

Standard immunohistochemical techniques and laser scanning confocal microscopy were used to assess changes in the abundance of Clara cell 10 kD protein (CC10) within granules and endoplasmic reticulum of rat nonciliated cells after the administration of the secretagogue pilocarpine. Intracellular pools of CC10 were compared over time with time-matched controls at two airway levels, proximal bronchi and terminal bronchioles. Three zones of reflectance intensity (high, medium, and low), corresponding to different densities of CC10 were used to quantify changes in CC10 levels. We observed a shift in the abundance of CC10 from endoplasmic reticulum to granules 30 min after injection of pilocarpine. In addition, the depletion of granule-based CC10 occurred earlier in bronchial cells (30 min) than in bronchiolar cells (60 min). We also noted that the density of CC10 within the two cellular compartments remained steady even though the levels of CC10 dropped due to degranulation. Following degranulation, CC10 levels returned to near-control levels. In the absence of pilocarpine, the percentage of cell volume occupied by the granule-based CC10 indicated that two varieties of nonciliated cells exist in bronchial airways. However, administration of pilocarpine abolishes this difference in CC10 abundance. Our results demonstrate that bronchial cells respond sooner to the secretagogue than do their bronchiolar counterparts and/or possess biosynthetic capabilities that are more easily overwhelmed than those of the terminal bronchiolar cells. These differences suggest that proximal nonciliated cells are distinct from distal nonciliated cells.

Quantitative comparison of intracellular concentration and volume of Clara cell 10 KD protein in rat bronchi and bronchioles based on laser scanning confocal microscopy.

We used an antibody to rat Clara cell 10 KD secretory protein (CC10) to compare the abundance of CC10 in non-ciliated epithelium of bronchioles and bronchi by immunohistochemistry and laser scanning confocal microscopy (LSCM) in the reflectance mode. Three zones of reflectance intensity (high, medium, low), directly related to CC10 content, were used to distinguish subcellular compartments of differing densities. Two major compartments contained CC10: granules and endoplasmic reticulum. Bronchiolar cell granules had relatively even reflectance, a high CC10 concentration (0.92 mg/ml), and occupied 7.5% of the cell volume. Bronchial cell granules were bi-zonal, lower in CC10 concentration (0.83 mg/ml), and occupied less cell volume (2.3%). Low-reflecting endoplasmic reticulum occupied a greater cell volume in bronchioles than in bronchi (32.8% and 22.1%, respectively). An intermediate-density zone consisted of endoplasmic reticulum close to granules. CC10 abundance, expressed as ng stored per unit surface area of epithelial basal lamina, was more than three times greater in bronchioles than in proximal bronchi (1.50 ng/mm2 vs 0.42 ng/mm2). These data demonstrate that the process of CC10 secretion differs markedly in non-ciliated epithelium of bronchi and bronchioles. Identifiable mucous-goblet cells did not contain CC10. The LSCM provides a method for quantifying intracellular CC10 pools in intact lung cells and has the potential for quantifying other proteins in subcellular compartments.

Differentiated bronchiolar epithelium in alveolar ducts of rats exposed to ozone for 20 months.

The effects of exposure to 1.0 ppm of ozone for twenty months were studied in male Fischer 344 rats. Light microscopic, morphometric, and immunohistological approaches were used to determine the distribution and degree of differentiation of ciliated and nonciliated bronchiolar epithelial (Clara) cells lining alveolar ducts of the central acinus, a primary target of ozone-induced lung injury. Alveolar duct pathways extending beyond the level of the most proximal alveolar outpocketing of terminal bronchioles were isolated in longitudinal profile. The distance that ciliated and nonciliated bronchiolar epithelial (Clara) cells projected down each alveolar duct pathway was determined by placing concentric arcs radiating outward from a single reference point at the level of the first alveolar outpocketing. A high degree of heterogeneity in the magnitude of bronchiolar epithelial cell extension into alveolar ducts was noted for each isolation and animal. Age-matched control animals also demonstrated variation in the degree of bronchiolar epithelial cell extension down alveolar ducts. In animals exposed to ozone, a striking similarity was noted by scanning electron microscopy in the surface characteristics of cells lining both terminal bronchioles and alveolar ducts. The presence of Clara cell secretory protein in cells of bronchioles and alveolar ducts was also detected immunohistochemically and visualized using confocal laser scanning microscopy in the reflectance mode. Well-differentiated ciliated and nonciliated bronchiolar epithelial cells were found lining alveolar septal tips and alveoli up to a depth of 1,000 mu into the pulmonary acinus after 20 months of exposure to ozone. No evidence of inflammation was present in alveolar ducts, suggesting that epithelial cell transformations in alveolar ducts is a natural consequence of lifetime exposures to oxidant gases.