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1.
Marie W. Wooten Victoria L. Rudick Michael J. Rudick M. Louise Higgins 《In vitro cellular & developmental biology. Plant》1985,21(4):207-215
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. 相似文献
2.
R C Read P Roberts N Munro A Rutman A Hastie T Shryock R Hall W McDonald-Gibson V Lund G Taylor 《Journal of applied physiology》1992,72(6):2271-2277
Pseudomonas aeruginosa rhamnolipid causes ciliostasis and cell membrane damage to rabbit tissue, is a secretagogue in cats, and inhibits epithelial ion transport in sheep tissue. It could therefore perturb mucociliary clearance. We have investigated the effect of rhamnolipid on mucociliary transport in the anesthetized guinea pig and guinea pig and human respiratory epithelium in vitro. Application of rhamnolipid to the guinea pig tracheal mucosa reduced tracheal mucus velocity (TMV) in vivo in a dose-dependent manner: a 10-microgram bolus caused cessation of TMV without recovery; a 5-micrograms bolus reduced TMV over a period of 2 h by 22.6% (P = 0.037); a 2.5-microgram bolus caused no overall changes in TMV. The ultrastructure of guinea pig tracheal epithelium exposed to 10 micrograms of rhamnolipid in vivo was normal. Application of 1,000 micrograms/ml rhamnolipid had no effect on the ciliary beat frequency (CBF) of guinea pig tracheal rings in vitro after 30 min, but 250 micrograms/ml stopped ciliary beating after 3 h. Treatment with 100 micrograms/ml rhamnolipid caused immediate slowing of the CBF (P less than 0.01) of human nasal brushings (n = 7), which was maintained for 4 h. Mono- and dirhamnolipid had equivalent effects. The CBF of human nasal turbinate organ culture was also slowed by 100 micrograms/ml rhamnolipid, but only after 4 h (CBF test, 9.87 +/- 0.41 Hz; control, 11.48 +/- 0.27 Hz; P less than 0.05, n = 6), and there was subsequent recovery by 14 h.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
3.
Studies on ciliary dyskinesia factor in cystic fibrosis. IV. Its possible identification as anaphylatoxin (C3a)-IgG complex 总被引:7,自引:0,他引:7
Presumptive evidence indicates that the substance responsible for the pathophysiologic conditions of cystic fibrosis (CF) is a complement component, C3a, also known as anaphylatoxin. The known biochemical and physiological properties of C3a, together with the behavior of this purified substance when associated with IgG in our tracheal bioassay, compare favorably with those of molecular species which we have separated from sera and cell cultures of CF homozygotes and heterozygotes. Sera from normal healthy subjects, previously inactive by bioassay for ciliary dyskinesia factor (CDF), were converted to a CDF positive state by incubation with epsilon-amino-caproic-acid (EACA). EACA is a known inhibitor of the carboxypeptidase-B-like anaphylatoxin inactivator, and is the methof of choice for accumulating anaphylatoxins in normal blood. Incubation of EACA-treated normal sera and two fresh CDF-positive CF sera with carboxypeptidase-B produced reversion in all instances to a CDF negative, normal state. It is proposed that anaphylatoxin inactivator, a carboxypeptidase-B-like enzyme, is defective or deficient in cystic fibrosis and that this deficiency is the primary gene defect. 相似文献
4.
Cystic Fibrosis serum or its isolated component IgG fraction and calcium ionophore A23187 all produced a quantitatively greater increase of mucus glycoprotein secretion in the rabbit tracheal epithelium than did control serum or its isolated component IgG fraction. These values were determined by dry weight secretion per gram of tissue and on subsequent sialic acid content of secretions. This demonstrable increase in mucus production represents a measurable difference in the functioning of the cultured mucociliary apparatus due to the influence of cystic fibrosis serum. 相似文献
5.
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. 相似文献
6.
Byron K. Wolffing Michael G. Gabridge 《In vitro cellular & developmental biology. Plant》1979,15(4):308-314
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. 相似文献
7.
Intracellular levels of adenosine triphosphate in hamster trachea organ cultures exposed to Mycoplasma pneumoniae cells or membranes 总被引:11,自引:0,他引:11
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. 相似文献
8.
B H Bowman B J Lankford G M Fuller S D Carson A Kurosky D R Barnett 《Biochemical and biophysical research communications》1975,64(4):1310-1315
A small polypeptide within the molecular weight range of 6,000 to 11,000 was found in immunoglobulin fractions from sera of cystic fibrosis homozygotes and heterozygotes. This factor appears to be responsible for interfering with ciliary activity in oyster gills. Since it can be dissociated from IgG without reductive cleavage it must be bound in a non-covalent manner. After its dissociation the IgG fractions from cystic fibrosis sera no longer inhibited ciliary activity. This finding explains the differences previously observed in the molecular weights of the ciliary inhibitor synthesized by cultured cells and that of sera from cystic fibrosis genotypes. 相似文献
9.
Functional and ultrastructural effects of nontypeable haemophilus influenzae in a hamster trachea organ culture system 总被引:1,自引:0,他引:1
Joseph M. Mylotte Richard R. Stack Timothy F. Murphy John Asirwatham Michael A. Apicella 《In vitro cellular & developmental biology. Plant》1985,21(10):575-582
Summary A hamster trachea organ culture system was utilized to evaluate quantitatively the effects of a strain of nontypeableHaemophilus influenzae (NTHI) and culture supernatants of the same strain on ciliary activity. Tracheal explants were maintained in organ culture
for 96 to 144 h and ciliary activity was observed daily with an inverted microscope. Explants continuously exposed to a strain
of NTHI had a progressive decline in ciliary activity which was significantly lower than uninfected controls evaluated concomitantly
by 48 h of exposure and thereafter. Histologic studies revealed a progressive degeneration of mucosal cells and exfoliation
of ciliated cells. Scanning electron microscopy showed little adherence of NTHI to the mucosal surface. Sterile broth cultures
of NTHI and supernatants of organ cultures infected with the same NTHI strain had no adverse effect on ciliary activity. Infected
tracheal explants treated with ampicillin 24, 48, or 72 h after continuous bacterial challenge had no significant decline
in ciliary activity compared to controls. The lack of adherence and the histologic changes observed when hamster trachea cultures
were infected with NTHI suggested a toxin might mediate the damage observed. Broth and organ culture supernatants, however,
produced no damage. Therefore, further studies are needed to determine the role, if any, of a toxin in the production of damage
to hamster tracheal explants by NTHI.
This work was supported by a Merit Review grant from the Veterans Administration and by Grant AI-19641 from the National Institute
of Allergy and Infectious Diseases. 相似文献
10.
Infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent 总被引:1,自引:0,他引:1
Avian Infectious bronchitis virus (IBV) is a coronavirus that infects chickens via the respiratory epithelium as primary target cells. The binding of coronaviruses to the cell surface is mediated by the viral surface protein S. Recently we demonstrated that alpha2,3-linked sialic acid serves as a receptor determinant for IBV on Vero cells and primary chicken embryo kidney cells. Here we analyze the importance of the sialic acid binding activity for the infection of tracheal organ cultures (TOCs) by different IBV strains. Our results show that alpha2,3-linked sialic acid also serves as a receptor determinant on chicken TOCs. Infection of TOCs by IBV results in ciliostasis. Desialylation induced by neuraminidase treatment of tracheal organ cultures prior to infection by IBV delayed the ciliostatic effect or resulted in partial loss of ciliary activity. This effect was observed with both respiratory and nephropathogenic strains. Inhibition of ciliostasis was also observed when TOCs were pretreated with an alpha2,3-specific neuraminidase. Analysis of the tracheal epithelium for reactivity with lectins revealed that the susceptible cells in the epithelium abundantly express alpha2,3-linked sialic acid. These results indicate that alpha2,3-linked sialic acid plays an important role for infection of the respiratory epithelium by IBV. 相似文献
11.
The cystic fibrosis ciliary inhibitor (CFCI) has been fractionated from plasma of cystic fibrosis (CF) homozygotes and from the media of cultured fibroblasts derived from CF homozygotes. Plasma and fibroblast media from normal controls have been fractionated in an identical manner. Fractions from plasma and fibroblast culture media that demonstrate ciliary inhibitory activity contain several proteins in a molecular weight range of approximately 5,000-11,000. These proteins have been partially characterized by immunochemical analysis with antisera to 33 human serum proteins. Immunological determinants of albumin, C3 (but not C3a), C4, C5, alpha1-lipoprotein, beta-lipoprotein, beta2-microglobulin and immunoglobulin light chains have been detected by hemagglutination in fractions of CF plasma that inhibited ciliary activity and in analogous fractions from normal sera. None of the proteins were detected in media of cultured fibroblasts from either genotype. Since the same proteins and protein fragments were identified in both CF and normal plasma fractions, and were not detected in CF fibroblast media, it appears that none of these proteins can be identified as the CFCI. Identification of these proteins will permit further purification of the CFCI by immunochemical methods. 相似文献
12.
Effect of prostaglandin F2 alpha on pulmonary rapidly-adapting-receptors in the guinea pig 总被引:1,自引:0,他引:1
Pulmonary rapidly-adapting-receptors ( RARs ) are sensory nerve endings whose afferent fibers can be recorded in the vagus nerve. RARs may play a role in reflex bronchoconstriction as seen in anaphylaxis. They can be stimulated by chemical mediators of anaphylaxis, such as prostaglandin F2 alpha (PGF2 alpha). PGF2 alpha aerosol was administered to saline and bovine serum albumin (BSA)-treated guinea pigs while recording the activity of RARs . PGF2 alpha (250 micrograms/ml) given for 7-13 minutes increased both tracheal pressure and nerve activity over that produced by saline exposure in untreated guinea pigs. PGF2 alpha administered for three minutes (5-100 micrograms/ml) increased RAR nerve activity in a dose-related manner in the first five minutes of the experiment only in the BSA treated guinea pigs. Since changes in tracheal pressure did not show a significant dose-response relationship, the RARs responding to PGF2 alpha seemed to be stimulated by a direct mechanism. No correlation was shown between tracheal pressure and RAR nerve activity during PGF2 alpha treatment. Whereas, a significant correlation was found between tracheal pressure and RAR nerve activity during histamine aerosol treatment (r = 0.985). Histamine aerosol (1 to 1000 micrograms/ml, 3 min.) increased intratracheal pressure for 3 out of 4 doses. RAR nerve activity increased significantly only at the highest dose. Therefore, a possible direct effect of PGF2 alpha upon RARs exists while the effect of histamine seems dependent upon changes in airway pressure in the guinea pig. 相似文献
13.
Michael G. Gabridge Robert B. Polisky 《In vitro cellular & developmental biology. Plant》1977,13(8):510-516
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. 相似文献
14.
Membrane function was examined in cultured cells from cystic fibrosis patients by assaying several enzymes on intact skin fibroblasts attached to culture dishes. This technique required few cells and minimized disruption of cellular organization. Comparison of enzyme activities of intact and broken cells showed that 12% of total glucose-6-phosphate dehydrogenase, a cytoplasmic enzyme, was measurable using intact cells, while all adenosine monophosphatase was measurable using intact cells. Alkaline paranitrophenylphosphatase activity was divided between the cell surface and interior. Substrate competition experiments indicated that substrate specificities for adenosine monophosphatase and paranitrophenylphosphatase activities were different. Adenosine monophosphatase activities of 2 control and 2 cystic fibrosis strains fluctuated similarly during the cell culture cycle. The apparent Km values relative to adenosine monophosphate were similar in all strains. A chromatographic fraction of serum from a cystic fibrosis patient that was inhibitory to oyster ciliary activity had no effect on adenosine monophosphatase activity of normal fibroblasts. Furthermore, fractions of media from cystic fibrosis homozygote and heterozygote fibroblast cultures were not inhibitory to adenosine monophosphatase activities of intact normal fibroblasts or of part iculate fractions prepared from them. In light of previous studies that showed that factors from cystic fibrosis serum of culture medium disrupted specific membrane activities, it is proposed that the cystic fibrosis factor interacts with the plasma membrane, interfering most conspicuously with the protein functions that are sensitive to changes in their membrane environment. 相似文献
15.
D Junglee A Penketh A Katrak M E Hodson J C Batten P Dandona 《BMJ (Clinical research ed.)》1983,286(6379):1693-1694
Patients with cystic fibrosis have been found to have abnormal serum concentrations of immunoreactive trypsin and abnormal activities of pancreatic isoamylase. A study was undertaken to discover whether activity of pancreatic lipase is also altered in cystic fibrosis. Serum from 23 patients with cystic fibrosis was assayed for immunoreactive trypsin and pancreatic lipase. Median serum pancreatic lipase activity was significantly lower in patients with cystic fibrosis than in controls, as was immunoreactive trypsin concentration (p less than 0.0001). Some patients had supranormal lipase concentrations but these were not always associated with absence of malabsorption. Serum pancreatic lipase activity is considerably changed in cystic fibrosis. 相似文献
16.
In the present work, the effect of the alcoholic extract of the leaves of Aegle marmelos Corr. on guinea pig isolated ileum and tracheal chain was investigated, as this plant is used traditionally to treat asthma and related afflictions. These effects were investigated using the isolated organ bath method. 1 mg/ml and 2 mg/ml doses of the alcoholic extract of this plant produced a positive relaxant effect in isolated guinea pig ileum and tracheal chain, respectively. In addition, they antagonized the contractions, which are produced by histamine. Because the alcoholic extracts elicited the antagonistic effect against histamine and also relaxed the histamine-induced contractions, it can be concluded that relaxations induced by A. marmelos in both guinea pig ileum and tracheal chain were due to the depression of H1-receptors. Since we observed a complete relaxation of the guinea pig ileum and tracheal chain produced by the extract, we investigated its antagonistic effect against histamine. These results were due to the presence of one or more anti-histaminic constituents present in the alcoholic extract of this plant, therefore supporting to the traditional use of A. marmelos in asthmatic complaints. 相似文献
17.
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. 相似文献
18.
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. 相似文献
19.
Development of a sensitive, easily performed, reliable test would be an important advance in detecting cystic fibrosis, improving genetic counselling and providing early effective treatment. The sweat chloride test, which is reliable in diagnosis, is technically too difficult for a screening program, and only reliably detects homozygotes. In contrast, the meconium test for detecting homozygote newborns is simple, inexpensive, reasonably specific but its general application has yet to be evaluated. Detection of serum components is the basis of two new tests to distinguish patients with cystic fibrosis and carriers. The effect of these serum components on ciliary activity is the principle of one test, an extremely difficult procedure that is subjective and lacks sufficient specificity for routine use. The second test, in which serum components are separated by isoelectric focusing, may provide an objective biochemical means of detecting both homozygotes and heterozygotes. 相似文献
20.
T Kaizu S A Lyons C E Cross M D Jennings J A Last 《Comparative biochemistry and physiology. B, Comparative biochemistry》1979,62(3):195-200
1. The acidic and neutral glycoproteins secreted by cultured tracheal explants from pigs, sheep, rats, mice, monkeys, guinea pigs, dogs and chickens were purified and fractionated by column chromatography on DEAE-cellulose and by electrophoresis on cellulose acetate. 2. The ratios of acidic to neutral mucus glycoproteins were compared for the above animals with that of mucus glycoproteins secreted by cultured human bronchi. 3. The observed ratios of acidic to neutral glycoproteins ranged from 4.0 (mouse) to 7.2 (chicken and pig) from cultured tracheae; secreted human bronchial mucus had a ratio of 2.7. 4. The ratio of acidic to neutral glycoproteins secreted by tracheal explants varied with duration of incubation of the trachea in culture. 相似文献