Intracellular levels of adenosine triphosphate in hamster trachea organ cultures exposed to Mycoplasma pneumoniae cells or membranes

The amount of adenosine triphosphate (ATP) in hamster trachea organ cultures was determined with a technique based on light emission from a luciferin/luciferase/ATP reaction. The amount of ATP, expressed as ng per mg dry weight, was consistent in tracheal explants prepared from various animals and changed negligibly when explants were cultivated in vitro for several days. The amount of ATP was related directly to cellular activity and integrity in the epithelium since inactivation by heat or freeze-thaw rapidly depleted measurable ATP, and ciliary activity and ATP content were related directly. When tracheal explants were infected with 10(5) to 10(7) CFU of virulent Mycoplasma pneumoniae cells, both ciliary activity and ATP content in the tissue dropped dramatically after approximately 5 to 8 days (up to 85% and 60% decreases, respectively). Exposure of explants to 50 to 200 microgram per ml of purified M. pneumoniae membranes also caused significant decreases in ciliary activity and ATP. When explants were infected with attenuated or nonvirulent mycoplasmas, ciliary activity was only slightly decreased, while ATP values often rose slightly. The technology associated with the determination of ATP levels in tracheal explants should prove useful as a new, objective, analytical approach to cell viability in organ cultures.     

Intracellular levels of adenosine triphosphate in hamster trachea organ cultures exposed toMycoplasma pneumoniae cells or membranes

Summary The amount of adenosine triphosphate (ATP) in hamster trachea organ cultures was determined with a technique based on light emission from a luciferin/luciferase/ATP reaction. The amount of ATP, expressed as ng per mg dry weight, was consistent in tracheal explants prepared from various animals and changed negligibly when explants were cultivated in vitro for several days. The amount of ATP was related directly to cellular activity and integrity in the epithelium since inactivation by heat or freeze-thaw rapidly depleted measurable ATP, and ciliary activity and ATP content were related directly. When tracheal explants were infected with 10⁵ to 10⁷ CFU of virulentMycoplasma pneumoniae cells, both ciliary activity and ATP content in the tissue dropped dramatically after approximately 5 to 8 days (up to 85% and 60% decreases, respectively). Exposure of explants to 50 to 200 μg per ml of purifiedM. pneumoniae membranes also caused significant decreases in ciliary activity and ATP. When explants were infected with attenuated or nonvirulent mycoplasmas, ciliary activity was only slightly decreased, while ATP values often rose slightly. The technology associated with the determination of ATP levels in tracheal explants should prove useful as a new, objective, analytical approach to cell viability in organ cultures. This investigation was supported in part by the National Institutes of Health (PHS Grant AI 12559), by a Biomedical Sciences Support Grant made to the University of Illinois School of Life Sciences, and by the University Research Board.     

Effect of in vitro cultivation andMycoplasma pneumoniae infection on intracellular cyclic AMP levels in hamster tracheal organ cultures

Summary Exogenous cyclic AMP and dibutyryl cyclic AMP decreased the relative ciliary activity values of tracheal organ cultures. In contrast, theophylline and cholera toxin were not ciliostatic. The use of a radioimmunoassay for cyclic AMP indicated that all of the tested substances increased intracellular cyclic AMP levels to some extent (from 3-fold for cholera toxin to almost 40-fold for dibutyryl cyclic AMP). Physical inactivation of explants by either freeze-thaw or heat destroyed all ciliary activity and greatly decreased intracellular cyclic AMP levels. Cyclic AMP levels of explants remained relatively constant during in vitro cultivation. Three strains ofMycoplasma pneumoniae were found to contain extremely low amounts of cyclic AMP. Infection of tracheal explants produced a significant decrease in relative ciliary activity, but only a slight decline in organ-culture cyclic AMP levels. This study was supported in part by Grant AI 12559 from the National Institutes of Health. The supply of cholera toxin from Dr. R. A. Finkelstein is most appreciated as are the assistance and advice of J. A. Engelhardt and Y. D. B. Stahl.     

Effect of in vitro cultivation and Mycoplasma pneumoniae infection on intracellular cyclic AMP levels in hamster tracheal organ cultures.

Exogenous cyclic AMP and dibutyryl cyclic AMP decreased the relative ciliary activity values of tracheal organ cultures. In contrast, theophylline and cholera toxin were not ciliostatic. The use of a radioimmunoassay for cyclic AMP indicated that all of the tested substances increased intracellular cyclic AMP levels to some extent (from 3-fold for cholera toxin to almost 40-fold for dibutyryl cyclic AMP). Physical inactivation of explants by either freeze-thaw or heat destroyed all ciliary activity and greatly decreased intracellular cyclic AMP levels. Cyclic AMP levels of explants remained relatively constant during in vitro cultivation. Three strains of Mycoplasma pneumoniae were found to contain extremely low amounts of cyclic AMP. Infection of tracheal explants produced a significant decrease in relative ciliary activity, but only a slight decline in organ-culture cyclic AMP levels.     

A tracheal culture model of respiratory tract infection withPseudomonas Aeruginosa

Summary The pathogenesis ofPseudomonas aeruginosa for the respiratory tract has been examined using hamster tracheal organ cultures. Tracheal rings prepared from male Syrian hamsters, strain LSH/LAK, were infected withP. aeruginosa for 4 h and processed at 4-h intervals for 24 h for examination by light- and electron microscopy. Tissue destruction was observed within 8 h after infection with 10⁸ colony-forming units (cfu)/ml and within 12 h after infection with 10⁴ or 10⁶ cfu/ml. Ciliated cells that contained abnormal subcellular organelles were expelled from the epithelium. By 20 h the epithelial borders were composed primarily of nonciliated cells. Transmission- and scanning electron microscopy revealed details of the cellular destruction and attachment ofP. aeruginosa to the ciliated epithelium.Pseudomonas aeruginosa causes a rapid destruction of the epithelium of hamster trachea in cultures. Hamster tracheal organ cultures have been shown to be useful in studying the pathogenesis ofP. aeruginosa for the respiratory tract. This work was supported by Grants G-430B and G-431B from the Cystic Fibrosis Foundation.     

A whole-organ perfusion model of bordetella pertussis adherence to mouse tracheal epithelium

Summary A whole-organ perfusion system was used to culture tracheas from adult Swiss mice and test this system's adaptability for use in adherence assays for virulentBordetella pertussis. Culture medium and bacterial suspensions flowed readily through the tracheal lumen, ciliary activity was maintained throughout the culture period, and scanning electron microscopy revealed retention of normal surface morphology. The number of adherent colony-forming units (cfu) per trachea was determined for all threeBordetella species every 30 min over a 3.5-h incubation period and the resultant adherence patterns were reproducible. Adherent cfu were dependent on the concentration of microorganisms in the infecting inoculum.Bordetella pertussis did not demonstrate a preferential adherence to either the dorsal or ventral surface of the tracheal epithelium nor did it demonstrate a preference for adherence to the laryngeal or bronchial end of the trachea. Static growth conditions did alter the adherence pattern ofB. pertussis from that observed when the organism was grown with constant agitation. This work was presented in part at the 1984 annual meeting of the American Society for Microbiology, St. Louis, MO.     

In vitro metabolism and biological activity of all-trans-retinoic acid and its metabolites in hamster trachea

The in vitro metabolism of all-trans-[11,12-3h]retinoic acid to several more polar compounds has been demonstrated in a hamster tracheal organ culture system. The production of these metabolites is dependent on the presence of tissue. The physiological significance of these compounds is shown by the cochromatography of several of the in vitro formed metabolites synthesized from [carboxy-14C]retinoic acid with metabolites isolated from the intestine and urine of hamsters previously injected with 0.1 to 1.5 microgram of [3H]retinoic acid. One of the metabolites shows about one-tenth the biological activity of all-trans-retinoic acid when tested in a hamster tracheal organ culture assay. This biologically active metabolite is converted by the hamster trachea in vitro to a biologically inactive metabolite.     

Cytopathic effects of the pathogenic Neisseria. Studies using human fallopian tube organ cultures and human nasopharyngeal organ cultures

Infection of mucosal surfaces by N. gonorrhoeae and N. meningitidis may result in inflammation indicating potential injury to host cells. We used human fallopian tube organ cultures (FTOC) and human nasopharyngeal organ cultures (NPOC) to study the mechanisms by which gonococci and meningococci damage human mucosal surfaces. Early in the course of FTOC infected with gonococci and NPOC infected with meningococci, damage was most apparent to ciliary activity. Loss of ciliary activity was accompanied by sloughing of ciliated cells. The damage to ciliated cells was not associated with attachment of gonococci or meningococci to these cells or the presence of organisms within ciliated cells. Infection with the commensal N. subflava did not result in significant damage to human FTOC or NPOC ciliary activity. LPS appears to be a major toxin of gonococci for human FTOC ciliated cells. Gonococcal peptidoglycan fragments also damage FTOC ciliary activity. Both piliated (P+) and nonpiliated (P-) gonococci and meningococci damage FTOC and NPOC ciliary activity, but P+ organisms damage ciliary activity more rapidly than P- organisms. Damage to FTOC ciliated cells was produced by <10 g/ml of purified gonococcal and meningococcal LPS. By 1–2h after exposure to LPS, vesicles containing LPS were distributed throughout the cytoplasm of ciliated cells. Polymyxin B neutralized LPS-induced damage, suggesting that the lipid A portion of LPS was the toxic moiety. In contrast, purified gonococcal and meningococcal LPS at 100 g/ml did not damage human NPOC or FTOC from rabbits, pigs and cows. These studies indicate that N. gonorrhoeae and possibly N. meningitidis damage ciliated epithelial celsl indirectly by release of toxins from the organisms. The differences in susceptibility of FTOC and NPOC to LPS may suggest changes in density of receptors for LPS and may help explain variation in severity of gonococcal and meningococcal interactions at different human mucosal surfaces.     

Large-Quantity Production of Chicken Embryo Tracheal Organ Cultures and Use in Virus and Mycoplasma Studies

Chicken tracheal organ cultures were made from embryos which were 19 to 20 days old. Transversely cut rings of trachea were placed in screw-capped tissue-culture tubes with Eagle's-N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) medium and incubated in roller drums. The method had advantages over other organ culture systems in that these cultures were prepared in numbers similar to conventional tissue cultures, ciliary activity was quickly and accurately evaluated, and contamination occurred less frequently than with organ cultures in petri dishes. Ciliary activity persisted for at least 1 month when the medium was changed at 5-to 7-day intervals and for 10 to 15 days without a change. Infectious bronchitis virus stopped ciliary movement, and this effect was used as a basis for titrating the virus and for determining the neutralizing capacity of immune mouse ascitic fluid. Twenty-four Mycoplasma strains were tested. Organisms of 17 strains, both avian and mammalian, multiplied in the organ cultures, and 7 strains, belonging to the species M. gallisepticum and M. mycoides var. capri, inhibited ciliary activity.     

Evidence of DNA repair in organ cultures of hamster tracheal epithelium following exposure to gas phase singlet oxygen

Autoradiographic identification of unscheduled DNA synthesis (UDS) in short-term organ culture of hamster tracheal epithelium has been used as a predictive test for mutagenic and/or carcinogenic compounds. Tracheal explants were treated for 2 h with singlet delta oxygen plus [3H]thymidine. Silver grains over the nuclei of epithelial cells from the superficial layer of the mucosa were observed, indicating UDS. Control cultures, exposed to the gas phase without singlet oxygen, failed to elicit UDS.     

The effects of proteins secreted by fibroblasts from patients with cystic fibrosis on hamster tracheal explants

Summary Hamster tracheal explants have been used to assay for mucosecretory activity in media taken from cultures of fibroblasts isolated from patients with cystic fibrosis (CF). Cystic fibrosis and normal sera were first used to establish optimal conditions for mucus release in the hamster tracheal ring assay. Unless protein levels were maintained at 5% serum concentration or greater there was loss of cilia, nonspecific mucus accumulation, and extensive epithelial damage to the luminal surface. Likewise, it was shown that exposure of the explants to unconcentrated conditioned media from CF (GM 770, 768, 1348, 142) or normal (GM 3349, 38) cultured fibroblasts for 1, 6, or 12 h resulted in the same type of damage and this was due to low protein levels. When the protein concentration of the conditioned media was increased with fetal bovine serum, the morphological integrity of the explants was maintained, demonstrating that there was no apparent difference between CF and normal fibroblast-secreted proteins in ability to induce mucus release. The ciliary inhibitory capacity of CF serum-derived or fibroblast-derived factor had been reported to require IgG for activity. However, addition of IgG to high molecular weight (VoP10) or low molecular weight (VeP10) secreted proteins had no apparent effect on stimulating secretion. In conclusion, it is possible that CF fibroblasts do not secrete a protein that has the mucostimulatory effect and thus these cells may not be suitable for studying the CF-related activity.     

Adherence of clinical isolates ofPseudomonas aeruginosa to hamster trachael epithelium in vitro

The adherence to hamster tracheal epithelium, of mucoid and nonmucoid clinical isolates ofPseudomonas aeruginosa from cystic fibrosis patients, was studied using tracheal organ cultures. Tracheal cultures were infected with 10⁷ colony-forming units per ml of either mucoid or nonmucoid clinical isolates ofP aeruginosa. The tracheal explants were rinsed at various time intervals to remove nonadherent bacteria, fixed, and prepared for transmission-and scanning-electron microscopy. Mucoid isolates were seen adhering to the ciliated epithelium as early as 4 h after initiation of infection, whereas nonmucoid isolates were only observed adhering at 6 to 8 h after infection. Mucoid organisms were found as clusters of bacteria embedded in an extensive extracellular matrix. The nonmucoid isolates were generally found as single organisms with no evidence of an extracellular matrix. These results suggest that the prevalence of mucoid isolates ofP. aeruginosa in cystic fibrosis may be due to adherent properties of the mucoid organism.     

Cellular retinoic acid-binding protein and its relationship to the biological activity of four synthetic retinoids in hamster tracheal organ culture

Summary The mechanism of action of retinoid in reversing keratinization in hamster trachea is yet unknown. The purpose of this study was to determine if cellular retinoic acid binding protein (CRABP) is present in tracheal epithelium following incubation in serum-free, vitamin A-deficient culture medium for 10 days, and if the effectiveness of a retinoid in reversing keratinization in organ culture is correlated with its ability to compete for CRABP sites. The cytosol prepared from tracheal cultures contained CRABP at a concentration of 2.61 pmoles per mg protein. Of the four retinoids with carboxyl end group selected for the study, two of the biological active retinoids competed for the CRABP sites. However, no correlation was observed between the biological activity of the inactive retinoids and their ability to associate with the CRABP sites. These results indicate that even though the action of retinoid may be mediated by retinoid binding protein, it cannot be used as a sole predicator of retinoid response in hamster trachea. This investigation was supported by Contract N01-CP-31012 and U. S. P. H. Grants CA30512 and CA32428, which were awarded by the Division of Cancer Etiology, National Cancer Institute, DHHS. Editor's Statement Tracheal organ cultures provide a useful model for the study of epithelial differentiation and carcinogenesis. Much attention has been given to the action of retinoids in this process. Mehta et al. demonstrate a lack of correlation between biological activity and specific cytosolic binding of members of this class of compounds, pointing out the need for a more complete biochemical understanding of the mechanism of action and active forms of retinoids in this and other systems in vivo and in vitro. David W. Barnes     

Experimental pathogenicity evaluation of Mycoplasma canadense from bovine mastitis in vitro and in vivo laboratory models

Mycoplasma canadense, a clinical isolate from milk of a mastitic buffalo, was experimentally tested for its pathogenic potential in hamster tracheal ring and rabbit fallopian tube explant organ cultures (in vitro) and rat and rabbit mammary gland (in vivo) models. The activity percentage reduction in M. canadense infected hamster tracheal rings was 99.1% in comparison to 16.4% in control rings. Mycoplasma canadense, also induced complete ciliostasis at 11-day post-infection in rabbit fallopian tube explants. Histopathological lesions in these infected organ cultures were loss of cilia, desquamation or denudation of epithelium, infiltration of inflammatory cells and proliferation of macrophages as well as oedema in lamina propria. At the end of the experiments, M. canadense organisms were reisolated in pure colonies from the infected but not the control organ cultures. In the rat and rabbit mammary glands, M. canadense organisms persisted upto 6-day and 7-day postinfection, respectively and caused histopathological changes suggestive of subacute to chronic mastitis during the experimental period. The results indicate that the tested M. canadense clinical isolate was virulent.     

Preparation and long-term cultivation of porcine tracheal and lung organ cultures by alternate exposure to gaseous and liquid medium phases

Summary Conventional methods of organ culture have proved unsatisfactory for mammalian lung because of the rapid collapse of the tissue and the loss of its normal structure. In an effort to circumvent this problem and to provide a means for visualizing the cellular relationships throughout the culture period, respiratory organs consisting of trachea and lungs of fetal or hysterectomy-derived 1- to 4-week-old pigs were embedded in warm 3% Noble agar in phosphate buffer silicone solution and cooled to firmness. By use of a described cutting device, the respective organs were sliced into thin, 0.5- to 1.0-mm tracheal ring or lung explants. These organ sections then were cultured by exposure to alternate gaseous and liquid-medium phases by rotation (12 rev per hr) in sealed Leighton tubes fitted in a described rotator. In short-term culture experiments, explants were best maintained in a culture-support medium containing Eagle's minimal essential medium, 20% fetal bovine serum, 0.5% lactalbumin hydrolysate, and other supplements in a pH range of 6.5 to 8.2, and a NaCl concentration of 0.1m or less. By bright-field and scanning-electron microscopy, tracheal ring and lung explant cultures incubated for 2 months showed intact, uniform and active ciliated epithelial surfaces which compared favorably with those of fresh preparations. The lung cultures showed alveoli that remained expanded, and the cellular integrity of the tissues remained normal in appearance. This new method provides respiratory organs as continuous records with exceptional cellular clarity and readily available for histological processing. The organ cultures lend themselves well to pathogenesis studies in which subtle cellular changes or a sequence of changes induced in pulmonary tissues are difficult to observe in the host.     

Preparation and long-term cultivation of porcine tracheal and lung organ cultures by alternate exposure to gaseous and liquid medium phases.

Conventional methods of organ culture have proved unsatisfactory for mammalian lung because of the rapid collapse of the tissue and the loss of its normal structure. In an effort to circumvent this problem and to provide a means for visualizing the cellular relationships throughout the culture period, respiratory organs consisting of trachea and lungs of fetal or hysterectomy-derived 1- to 4-week-old pigs were embedded in warm 3% Noble agar in phosphate buffer silicone solution and cooled to firmness. By use of a described cutting device, the respective organs were sliced into thin, 0.5- to 1.0-mm tracheal ring or lung explants. These organ sections then were cultured by exposure to alternate gaseous and liquid-medium phases by rotation (12 rev per hr) in sealed Leighton tubes fitted in a described rotator. In short-,erm culture experiments, explants were best maintained in a culture-support medium containing Eagle's minimal essential medium, 20% fetal bovine serum, 0.5% lactalbumin hydrolysate, and other supplements in a pH range of 6.5 to 8.2, and a NaCl concentration of 0.1 M or less. By bright-field and scanning-electron microscopy, tracheal ring and lung explant cultures incubated for 2 months showed intact, uniform and active ciliated epithelial surfaces which compared favorably with those of fresh preparations. The lung cultures showed alveoli that remained expanded, and the cellular integrity of the tissues remained normal in appearance. This new method provides respiratory organs as continuous records with exceptional cellular clarity and readily available for histological processing. The organ cultures lend themselves well to pathogenesis studies in which subtle cellualr changes or a sequence of changes induced in pulmonary tissues are difficult to observe in the host.     

Mycoplasma pneumoniae infection of intact guinea pig tracheas cultured in a unique matrix-embed/perfusion system

Summary A new method for the in vitro culture of entire, intact tracheas from adult guinea pigs is described. Matrix-embed/perfusion (MEP) culture is based on an immobilization of the tissue in nutrient agar. The tubular piece of agar-embedded organ was contained in a special perfusion block with two wells for liquid medium at either end. When incubated on a rocker platform, liquid medium flows through the trachea and supplies oxygen and nutrients. In this configuration, tracheas maintain near-normal metabolism (ATP content and dehydrogenase activity), structure (as determined by light and electron microscopy), and function (ciliary motion). Tissues could be maintained in vitro in a normal state for at least 4 wk, with reduced ciliary motion and cell metabolism detectable for at least 6 wk. Agarembedded tissues from the MEP cultures were nearly identical to those cultivated with standard tracheal ring explant techniques. Tracheas in the MEP cultures were infected withMycoplasma pneumoniae. Attachment was neuraminidase-sensitive. Mycoplasma attachment was lowest on the epithelium along the dorsal ridge, but was uniform along the length of the trachea. Ciliostasis and cytonecrosis induced byM. pneumoniae was dose dependent. The matrix-embed/perfuse technique appears to have considerable potential for several types of in vitro studies on trachea or other tubular organs. This study was supported in part by USPHS Grants AI 12559 and AI 17795 from the National Institutes of Allergy and Infectious Diseases, and Grants HL 23806 and HL 26880 from the National Heart, Lung, and Blood Institute.     

Inhibition of ciliary activity in organ cultures of ferret trachea in reference to genetic and biological characters in influenza virus strains

As reported previously, attenuated stable inhibitor-resistant influenza viruses can be screened by a 50% ciliary activity inhibition test in ferret tracheal organ cultures. This test was further applied to a 5 attenuated cold-adapted influenza strains and to 11 strains with known a percentage of RNA-RNA hybridization with the parental A/PR/8/34 (HON1) virus strain. Again, with one exception, attenuated strains could be clearly differentiated from virulent ones. It was concluded that virulence of influenza strains for man can be detected using this test regardless of the techniques used to prepare attenuated variants. A preliminary screening of attenuated candidates for live influenza vaccines can be achieved with confidence on ferret tracheal organ cultures.     

Ciliary beating frequency of frog palate and rat trachea explants under continuous perfusion

Rat trachea and frog palate explants were continuously perfused for 2 hr with 199 tissue culture medium. The ciliary beating frequency of the frog palate exhibited fluctuations twice as large as compared to the rat trachea. The rat trachea model should be preferred to the frog palate for long-term studies on ciliary beat frequency.     

Assessment of the tracheal ring bioassay for detection of the cystic fibrosis serum factor

We have examined the effects of serum from cystic fibrosis patients, healthy human volunteers and from guinea pigs on ciliary activity of guinea pig tracheal ring explants after 48 hours in culture. Sera from 9 out of 10 cystic fibrosis patients produced ciliostasis. This is a significantly greater percentage than the 7 out of 21 serum samples from healthy volunteers that produced ciliostasis. Ninety-two percent of the explants in guinea pig serum had unaltered ciliary activity, illustrating the importance of intrinsic control in the bioassay design. These result suggest that the guinea pig tracheal ring bioassay may be of value as a means of identifying the presence of the ciliotoxic factor in cystic fibrosis serum for research use but is a poor discriminator for diagnostic purposes. Modification such as rinsing the tracheal mucosa with sterile medium and a new chamber for the microscopic observation of the tissue have simplified the assay.