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Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to irreversible loss of neurons, cognition and formation of abnormal protein aggregates. Rivastigmine, a reversible cholinesterase inhibitor used for the treatment of AD, undergoes extensive first-pass metabolism, thus limiting its absolute bioavailability to only 36% after 3-mg dose. Due to extreme aqueous solubility, rivastigmine shows poor penetration and lesser concentration in the brain thus requiring frequent oral dosing. This investigation was aimed to formulate microemulsion (ME) and mucoadhesive microemulsions (MMEs) of rivastigmine for nose to brain delivery and to compare percentage drug diffused for both systems using in-vitro and ex-vivo study. Rivastigmine-loaded ME and MMEs were prepared by titration method and characterized for drug content, globule size distribution, zeta potential, pH, viscosity and nasal ciliotoxicity study. Rivastigmine-loaded ME system containing 8% w/w Capmul MCM EP, 44% w/w Labrasol:Transcutol-P (1:1) and 48% w/w distilled water was formulated, whereas 0.3% w/w chitosan (CH) and cetyl trimethyl ammonium bromide (as mucoadhesive agents) were used to formulate MMEs, respectively. ME and MMEs formulations were transparent with drug content, globule size and zeta potential in the range of 98.59% to 99.43%, 53.8 nm to 55.4 nm and -2.73 mV to 6.52 mV, respectively. MME containing 0.3% w/w CH followed Higuchi model (r(2) = 0.9773) and showed highest diffusion coefficient. It was free from nasal ciliotoxicity and stable for three months. However, the potential of developed CH-based MME for nose to brain delivery of rivastigmine can only be established after in-vivo and biodistribution study.

Loss of function in either VHL or Nek1 leads to cyst formation in tissues, especially in kidneys. Whether there is a connection between pVHL and Nek1 regulation is unknown. Here, we report that the VHL protein (pVHL) may be a substrate of Nek1. While Nek1 can phosphorylate pVHL at multiple sites, the phosphorylation at serine-168 results in pVHL degradation. Nek1-mediated phosphorylation of pVHL does not significantly affect hypoxia-inducible factors (HIF), a known target of pVHL. However, non-phosphorylable pVHL reconstituted in VHL-deficient cells induces more stable cilia than wild-type VHL during serum stimulation and Nocodazole treatment. The results suggest a possible regulation of pVHL by Nek1 that may contribute to ciliary homeostasis and cystogenesis.

In order to improve brain uptake of nanoparticles following nasal administration, odorranalectin (OL), the smallest lectin with much less immunogenicity than other members of lectin family, was conjugated to the surface of poly (ethylene glycol)-poly (lactic-co-glycolic acid) (PEG-PLGA) nanoparticles (NP) in this study. The bioactivity of OL conjugated to the nanoparticles was verified by haemagglutination tests.Tissue distribution of OL-modified and unmodified nanoparticles (OL-NP and NP) was evaluated following intranasal (i.n.) administration by in vivo fluorescence imaging technique using DiR as a tracer, comparing with that of unmodified nanoparticles after intravenous (i.v.) injection. Besides, the nasal toxicity of OL-NP was evaluated on Calu-3 cell lines, toad palate and rat nasal mucosa.The results of TEM examination and dynamic light scattering showed a generally spherical shape of OL-NP with an average volume-based diameter around 90 nm. The haemagglutination test proved that OL retained its haemagglutination activity when conjugated to nanoparticles. The brain targeting indexes of NP and OL-NP following i.n. administration and NP following i.v. injection were 5.8, 11.6 and 0.08, respectively.Thus,i.n. administration demonstrated much better brain targeting efficiency than i.v. injection, and OL modification facilitated the nose-to-brain delivery of nanoparticles.Moreover, the toxicity assessment suggested good safety of OL-NP both in vitro and in vivo. In summary, odorranalectin-conjugated nanoparticle could be potentially used as a nose-to-brain drug delivery carrier for the treatment of CNS diseases.

Recent evidence suggests that fibrocystin/polyductin (FPC), polycystin-1 (PC1), and polycystin-2 (PC2) are all localized at the plasma membrane and the primary cilium, where PC1 and PC2 contribute to fluid flow sensation and may function in the same mechanotransduction pathways. To further define the exact subcellular localization of FPC, the protein product encoded by the PKHD1 gene responsible for autosomal recessive polycystic kidney disease (PKD) in humans, and whether FPC has direct and/or indirect cross talk with PC2, which, in turn, is pivotal for the pathogenesis of autosomal dominant PKD, we performed double immunostaining and coimmunoprecipitation as well as a microfluorimetry study of kidney tubular epithelial cells. FPC and PC2 are found to completely or partially colocalize at the plasma membrane and the primary cilium and can be reciprocally coimmunoprecipitated. Although incomplete removal of FPC by small interfering RNA and antibody 803 to intracellular epitopes of FPC did not abolish flow-induced intracellular calcium responses, antibody 804 to extracellular epitopes of FPC blocked cellular calcium responses to flow stimulation. These findings suggest that FPC and polycystins share, at least in part, a common mechanotransduction pathway.

Conclusions: The combination of N-chlorotaurine (NCT) and a corticosteroid seems to be a very promising substance for the local therapy of ENT infections. As it can be used without any preservatives, the effect on the ciliary beat frequency (CBF) is much less than that of products containing benzalkonium chloride (BAC). The in vitro results obtained in this study encourage us to perform clinical trials on this novel combination for intranasal application., Objective: To investigate the influence of a novel mixture of NCT and a corticosteroid [fluticasone propionate (FP)] on the CBF of human ciliated cells in vitro., Material and Methods: The study was designed as an in vitro study. CBF was measured by means of a photometric technique involving the combination of a light microscope, a photometer, a photographic multiplier and a computerized analyzing unit., Results: The combination of 1% NCT + 0.5 mg/ml FP decreased the CBF to 42.17% of its original value after 20 min. Treatment with BAC lowered the CBF depending on the concentration to 96.61% of its original value with 0.04 mg/ml, to 91.90% with 0.1 mg/ml, to 63.46% with 0.2 mg/ml and to 0% with 0.5 mg/ml. After rinsing in saline, the CBF of samples treated with 1% NCT + 0.5 mg/ml FP recovered to 68.93% of its original value.

PKD2, or polycystin 2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease, belongs to the transient receptor potential channel superfamily and has been shown to function as a nonselective cation channel in the plasma membrane. However, the mechanism of PKD2 activation remains elusive. We show that PKD2 overexpression increases epidermal growth factor (EGF)-induced inward currents in LLC-PK(1) kidney epithelial cells, while the knockdown of endogenous PKD2 by RNA interference or the expression of a pathogenic missense variant, PKD2-D511V, blunts the EGF-induced response. Pharmacological experiments indicate that the EGF-induced activation of PKD2 occurs independently of store depletion but requires the activity of phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K). Pipette infusion of purified phosphatidylinositol-4,5-bisphosphate (PIP(2)) suppresses the PKD2-mediated effect on EGF-induced conductance, while pipette infusion of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)) does not have any effect on this conductance. Overexpression of type Ialpha phosphatidylinositol-4-phosphate 5-kinase [PIP(5)Kalpha], which catalyzes the formation of PIP(2), suppresses EGF-induced currents. Biochemical experiments show that PKD2 physically interacts with PLC-gamma2 and EGF receptor (EGFR) in transfected HEK293T cells and colocalizes with EGFR and PIP(2) in the primary cilium of LLC-PK(1) cells. We propose that plasma membrane PKD2 is under negative regulation by PIP(2). EGF may reduce the threshold of PKD2 activation by mechanical and other stimuli by releasing it from PIP(2)-mediated inhibition.

Conclusions: These findings suggest that IL-1beta, increases ciliogenesis in NHMEE cells and that MUC8 protein may play a role in this process., Objectives: The morphology of the middle ear epithelium changes under various pathologic conditions. We compared the density of ciliated cells and the level of mucin gene 8 (MUC8) mRNA in the middle ear epithelial cells of mucoid otitis media (MOM) patients with those of normal controls. We also investigated the effect of IL-1beta on the ciliogenesis and expression of MUC8 mRNA in cultured normal human middle ear epithelial (NHMEE) cells. In addition, we localized the MUC8 protein in cultured NHMEE cells., Material and Methods: The surface morphology of NHMEE cells was examined using scanning electron microscopy. Reverse transcriptase polymerase chain reaction was performed to determine the level of MUC8 mRNA expression. After synthesis of MUC8 polyclonal antibody, we performed immunohistochemical staining., Results: The density of ciliated cells was increased in the middle ear epithelium of both MOM patients and IL-1beta-treated cultures. Similarly, the expression of MUC8 was increased in both cases. MUC8 antibody was primarily stained on the ciliated cells of NHMEE cells.

Objectives: Retinoic acid (RA) can prevent keratin formation and induce mucous differentiation in epithelia. In this study, we attempted to induce keratinizing squamous epithelium from human cholesteatoma epithelial (HCE) cells using an air-liquid interface (ALI) technique. We also examined the effect of RA on the phenotype of keratinizing HCE cells., Material and Methods: HCE cells were cultured in RA-free defined media at an ALI or in a submerged state. We examined the morphological differences between ALI and submerged cultures, and histologically investigated the changes of phenotype after RA treatment. We also determined the effect of RA on the mRNA expressions of the cornifin-alpha and mucin genes as indicators of squamous and mucous differentiation, respectively., Results: Using an ALI technique, we were able to differentiate HCE cells into a keratinizing squamous epithelium. When we treated the keratinizing HCE cells with RA, the morphological phenotype progressively changed into mucociliary epithelium. In addition, the expression of cornifin-alpha mRNA was suppressed, and the expressions of mucin gene 5AC (MUC5AC) and MUC5B mRNA increased progressively with RA treatment., Conclusion: We successfully developed a culturing system for keratinizing differentiation of HCE cells using the ALI technique in a defined medium. Our study also clearly showed that RA treatment led to mucociliary differentiation of HCE cells.

Ciliostimulation induced by various transmitters has been suggested to be mediated by the release of nitric oxide (NO). Freshly obtained adenoid tissue explants were pre-treated with the nitric oxide synthase (NOS) inhibitor N(G)-nitro L-arginine (L-NNA), to determine whether the ciliostimulators terbutaline, methacholine, substance P, and endothelin-1 require the release of NO to increase ciliary beat frequency (CBF) in vitro. The L-NNA pre-treatment affected the change in CBF induced by each of the ciliostimulators tested. To determine whether cyclic nucleotides also stimulate CBF by inducing the release of NO, an extra series of experiments were performed with dibutyryl cAMP and dibutyryl cGMP, and L-NNA pre-treatment. In contrast to the experiments with the various ciliostimulators, both dibutyryl cAMP and dibutyryl cGMP exerted ciliostimulatory effects that could not be inhibited by L-NNA. The present findings suggest that NO acts as an intermediate messenger in the ciliated epithelium in response to various transmitters and mediators. On the other hand, pre-treatment with the NOS inhibitor L-NNA did not affect ciliary response to the second messengers cAMP and cGMP, thus suggesting that NO dependent mechanisms do not constitute the sole pathway for the stimulation of ciliary function.

This study demonstrated the in vitro effect of diazepam on human ciliary beat frequency (CBF) from fifteen normal subjects using brush cytology. Tube A contained culture medium, tube B the diluent that diazepam intravenous injectate was carried in and culture medium (controls). Tube C, D and E contained, 0.4 mg/l, 4.0 mg/l and 40.0 mg/l of diazepam in culture medium respectively. The mean effective diazepam concentration in plasma is 0.4 mg/l. CBF was measured photometrically. The most vigorous cilia were measured in 5 areas taking 10 readings on each sample, 30 minutes and 1 hour after mixing. Standard deviation (SD) and confidence limits were calculated along with significance testing (p < 0.05) using the paired t-test and ANOVA. The mean of the CBF of tubes A and B were 13.44 (SD 2.65) and 13.67 (SD 2.48). There was a reduction of the CBF with increasing concentrations of diazepam at 30 minutes, 11.32 (SD 2.14), 10.29 (SD 1.58) and 4.14 (SD 1.57) tubes C. D and E respectively. There was a significant lowering in CBF of 17% (p < 0.01) of diazepam at the mean effective plasma level (tube C) when compared against the controls. CBF decreased over time and at 1 hour was 10.57 (SD, 1.36), 9.02 (SD, 1.39) and 3.58 (SD, 1.31) tubes C, D and E respectively. A proposed mechanism of altered intracellular calcium flux via the action diazepam on GABA receptors is described.

1. Hatching chicks were exposed to 10.9 ppm of formaldehyde vapour during the last 3 d of incubation in a commercial situation. 2. Samples from pre-selected regions of the entire respiratory tract, taken at 0, 6, 30 and 54 h post-vaporisation of formaldehyde, were examined by scanning electron microscopy for surface morphological features and associated pathological changes. 3. Clumping of cilia, blebs on the cilial wall, deciliation and exfoliation of the epithelium were all observed under the scanning electron microscope. 4. Lesions were more severe in chicks exposed for 54 h as compared to those exposed for 6 and 30 h. There were no regional differences in the lesions throughout the respiratory tract of all the chicks. 5. Limited observations suggest that the passage of formaldehyde vapour across the egg shell wall may influence the morphology of the respiratory lining of the developing embryo.

The effects of the fungicide benomyl and its breakdown products, carbendazim and butyl isocyanate, were examined on canine tracheal epithelial tissue in primary culture. Changes in ciliary frequencies were monitored with an optical spectrum analysis system. Serial dilutions of the test compounds were prepared in 100% corn oil and applied to the cell cultures for intervals up to 6 hours and frequencies measured at intervals of 15 minutes to 1 hour. Benomyl and butyl isocyanate caused concentration-dependent decreases in ciliary beat frequency. Benomyl at 300 micrograms/ml (3 mM) caused ciliostasis within 75 minutes of exposure. Butyl isocyanate at a molar concentration three times lower than benomyl (1 mM) caused a similar response, although within 30 minutes. The IBC50 for benomyl was 0.75 mM, while for butyl isocyanate it was 0.52 mM. Carbendazim caused a moderate decrease in frequency over a 6 hour exposure period. Benomyl caused moderate to severe swelling of the mitochondria of ciliated epithelial cells with other cell organelles appearing normal. Butyl isocyanate did not cause any noticeable effect on cell ultrastructure and the apparently low rate of penetration of carbendazim into cells made it impossible to obtain an effect which justified ultrastructural analysis. It appears, at least for benomyl and butyl isocyanate, that while the physiological effect of these two compounds (inhibition of ciliary beat) is the same, the sites of action in the cell may be different.

The aim of this study was to evaluate histologic changes after long-term administration of the topical nasal decongestants phenylephrine and oxymetazoline. Ninety healthy rabbits were divided into 3 groups for topical administration for 1 week, 2 weeks, and 4 weeks. Each group was subdivided into 3 subgroups by topical administration of phenylephrine. oxymetazoline, and physiologic saline as controls. Each study group thus consisted of 10 rabbits. Phenylephrine was administered by 2 puffs in the left nostril 5 times daily and oxymetazoline by 2 puffs twice a day using metered sprayers giving 0.10 ml in each puff with a dosage used in clinical practice. For statistical significance the Kolmogorov-Smirnov test was used. Light and electron microscopic examination were performed after obtaining nasal respiratory mucosa from the nasal septum of the rabbits. Administration of phenylephrine and oxymetazoline for more than 2 weeks caused histologic changes including ciliary loss, epithelial ulceration, inflammatory cell infiltration and subepithelial edema, and the changes were more pronounced with increasing administration duration of the decongestants. Ciliary loss was prominent in the 4-week phenylephrine and oxymetazoline groups. There were significantly severer changes in the morphologic variables in the decongestant groups compared with the control group according to administration duration (p < 0.05). Dilatation or vacuolization of mitochondria and endoplasmic reticula and vesicles ill the cytoplasm were observed in the 2- and 4-week phenylephrine groups as well as the 2- and 4-week oxymetazoline groups. Mild widening of the intercellular space was observed in the 4-week phenylephrine group. Purulent maxillary sinusitis developed in 6 of 10 rabbits treated for 4 weeks with phenylephrine. The results of this study suggest that the administration of decongestants may cause ciliary loss with subsequent inflammatory changes in the nasal respiratory mucosa.

Diminished mucociliary transport can occur in a type-I (Ig-E mediated) allergic reaction. We determined the effects of the allergy mediators prostaglandin D2 (PGD2) and prostaglandin E2 (PGE2) on the ciliary beat frequency (CBF) of human upper respiratory cilia in vitro. Human adenoid tissue was used as the source for ciliated epithelium. CBF was measured by a computerized photo-electric method. PGD2 (10(-8)-10(-5) M, n = 7) showed no statistically significant effect on CBF. PGE2 (10(-9)-10(-6) M, n = 10) caused a significant dose-dependent stimulation, with a maximum of 37% (ANOVA, p < 0.001). Thus prostaglandins D2 and E2 do not exert a direct negative influence on ciliary activity, which could account for a decrease in mucociliary transport. The stimulating effect of PGE2 may be relevant in promoting mucociliary clearance in vivo.

Nitrogen dioxide exposure-induced mucosal pathology of the guinea pig nose was studied. Guinea pigs were exposed to 3 ppm or 9 ppm of nitrogen dioxide for 6 h a day, 6 times weekly for 2 weeks, and sacrificed 24 h after the final exposure. Exposure to 3 ppm of nitrogen dioxide resulted in decreased ciliary activity and slight eosinophil accumulation on the epithelium and submucosal layer. More serious pathologies were observed in the nose of guinea pigs exposed to 9 ppm of nitrogen dioxide, including a more prominent eosinophil influx to the epithelium and epithelial injury due to activation of eosinophils. Epithelial damage induced by nitrogen dioxide could lead to hyperresponsiveness and may result in a prolonged allergic inflammation. Our study suggests that environmental nitrogen dioxide may contribute to hyperresponsiveness and thus be involved in the increased morbidity of allergic rhinitis.

To investigate the effects of sulfur dioxide (SO2) on olfactory epithelium, an experiment was performed with 56 mice from the same colony. Experimental animals were divided into three groups consisting of a 30-min exposure group (group 1), a 60-min exposure group (group 2), and a 120-min exposure group (group 3). The olfactory mucosa in these mice were studied by light microscopy immediately, and after 24 h, 48 h, or 72 h exposure to 20 ppm of SO2. Edema, loss of cilia, epithelial thinning, and epithelial desquamation in the olfactory epithelium were observed in groups 2 and 3. The basal lamina and the connective tissue were well preserved throughout the entire mucosa. Injuries to olfactory epithelium became severer with exposure time. These changes were further pronounced 24 h after exposure. Regenerated epithelia were not observed in any group. Scanning electron microscopic findings were consistent with light microscopic findings. Olfactory epithelial surface were consistent with light microscopic findings. Olfactory epithelial surface was sloughed off and revealed, underlining intact basal lamina. The results of this study suggest that early lesions of olfactory epithelium after exposure to SO2 may be primarily degenerative.

The effects of exogenous ATP and physical stimulation on ciliary beat depressed by lipopolysaccharide from Pseudomonas aeruginosa were studied in cell culture. Both the ciliary beat frequency (CBF) and the amplitude (CBA) of human respiratory cells in monolayer cell cultures were studied by using a differential interference microscope equipped with a high speed video. Both the ATP and the physiol stimulation stimulated temporarily the depressed CBF and CBA. In both groups the CBF and CBA increased in 1 min to the initial level and then gradually decreased to the level before the stimulation. The duration of the ATP stimulation on the CBF (10.1 +/- 1.8 min) and CBA (10.1 +/- 2.3 min) were significantly better than the duration of physical stimulation on CBF (8.0 +/- 1.8 min) and CBA (6.8 +/- 1.6 min). However, the areas under the CBF/CBA curves (from the beginning of stimulation until the initial level of CBF or CBA was reached again) did not differ significantly. After the removal of the bacterial toxin the CBF was restored to the initial level. In the present study the adenosine receptor antagonist could not prevent the ciliostimulative effect of exogenous ATP combined with the physical stimulation. Both exogenous ATP and physical stimulation have a clear but temporary stimulative effect on the ciliary beat depressed by bacterial toxin, ATP being slightly more effective. It seems that the effects of ATP are not mediated by adenosine receptors.

To study the detailed effects of exogenous adenosine triphosphate (ATP) on ciliary function, we used the differential interference microscope equipped with high speed video and evaluated ciliated cells from the human sinus mucosa in monolayer culture. With this system it was possible to evaluate all parts of ciliary motility with a minimum of interference from the mucous membrane, secretory cells and the autonomic nervous system. The best direct ciliostimulative effect of exogenous ATP on ciliary beat frequency (CBF) and ciliary beat amplitude (CBA) was observed at concentrations of ATP ranging from 10(-5) M to 10(-3) M. Exogenous ATP appeared to normalize the slightly damaged ciliary motility in groups with an initial CBF of 14 Hz or higher. When the CBF was less than 14 Hz, ATP produced an increase in CBF greater than 40%, an increase in CBA greater than 30%, but these did not reach the normal level in 5 min. The biggest increases: 59.9% in CBF and 40.7% in CBA were seen in the group with an initial CBF less than 9 Hz. In the cells with a low initial CBF and unsynchronized motility exogenous ATP increased the synchrony of ciliary movement together with an increase in CBF and CBA.

The movement of normal human nasal cilia was analyzed. Ciliary beat was recorded by means of a phase-contrast microscope equipped with a photodetector. The electrical signal was analyzed as follows: i) a power spectrum was calculated in order to measure ciliary beat frequency (CBF), ii) the beat cycles were averaged and the standard deviation of the waveform was computed to determine signal consistency (SC), and iii) the ratio of the duration of the smooth to that of the steep part of the cycles was measured. This was done under three different conditions: 1) normal or "initial", 2) after induction of "function loss", and 3) after "salbutamol stimulation". At "function loss," the cilia beat slower and with less harmony. CBF decreased from an average of 9.0 Hz in the "initial" condition to 5.8 Hz. SC decreased from an average of 5.7 to 1.9. After "salbutamol stimulation", average CBF was partially restored to 7.7 Hz, while average SC increased to 4.4. These findings indicate that in ciliary function studies, SC, as a measure for ciliary beat harmony, may be introduced alongside CBF as a second valuable parameter. In this study we were not able to identify different phases in the signal that might be used as a third parameter to indicate the effective and the recovery stroke.

The investigation sought to identify alterations of specific ciliated epithelial surface components after exposure to formaldehyde (HCHO) levels that decrease respiratory ciliary function. Bovine tracheae were reacted with an analog of N-hydroxysuccinimidobiotin to label epithelial surface-accessible components before exposure to HCHO. The tracheae were then exposed to 0, 16, 33, and 66 micrograms HCHO/cm2 epithelial surface for 30 min. Cilia were isolated from the epithelium, separated into membrane and internal axonemal portions, analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and either stained to detect proteins or transblotted to detect biotin-labeled components. Densitometric analysis of axoneme proteins showed a decrease in the total amount extracted with increased HCHO concentration, including axoneme-specific proteins, dynein, and tubulin. However, biotinylated proteins in the axoneme fractions proportionately increased. Membrane fractions showed little change in protein with increasing HCHO concentration. The majority of these is not biotin-labeled and thus not surface-accessible components. Biotinylated material in the membrane fractions showed a significant decrease with increased HCHO concentration, particularly of bands at 92, 98, and 105 kD. These data suggest that increasing HCHO exposure reduces both extractable ciliary axonemes and detergent-soluble surface components, possibly by stabilizing respiratory epithelial membranes. This process apparently strengthens association of certain surface components with the internal axoneme, thereby reducing subsequent solubilization in detergent.

Using computerized microphotometry, we studied the effects of prostaglandin E2 (PGE2), the prostacyclin analog Iloprost, and the thromboxane A2 analog U46619 on the ciliary beat frequency (CBF) of human nasal mucosa. Thirty-two normal subjects underwent nasal cytologic brushing of the inferior meatus to obtain ciliary samples, and a total of 5,640 ciliated cell clusters were analyzed. Each subject served as their own control. PGE2, 10(-10) to 10(-6) M, produced a significant dose dependent increase in CBF of up to 12% versus control. This increase was not significantly inhibited by the addition of the cyclooxygenase inhibitor indomethacin (10(-6) M). Iloprost, 10(-12) to 10(-6) M, also significantly increased CBF by 12.7% at 10(-8) M. This ciliostimulatory effect, however, was abolished by indomethacin. The thromboxane A2 analog, 10(-10) to 10(-6) M, did not significantly effect CBF. The present study demonstrates that a thromboxane A2 analog has no effect on ciliary motility, PGE2 has a direct ciliostimulatory effect, and a prostacyclin analog has a ciliostimulatory effect likely mediated by stimulation of the cyclooxygenase pathway within human cells.

The effect on the mucociliary (m.c.) wave frequency of sympathomimetic agonists and antagonists was studied in the maxillary sinus in anesthetized rabbits. The non-selective beta-adrenoceptor agonist isoprenaline (0.005-10 micrograms/kg i.a.) and the selective beta2-adrenoceptor agonist salbutamol (0.01-10 micrograms/kg, i.a.) induced a dose-dependent acceleration of the m.c. wave frequency, whereas the beta1-adrenoceptor agonist prenalterol (1-200 micrograms/kg, i.a.) did not influence the basal m.c. activity. The selective alpha1-adrenoceptor agonist phenylephrine (0.01-20 micrograms/kg, i.a.) and the selective alpha2-adrenoceptor agonist oxymetazoline (0.001-1 microgram/kg, i.a.) both induced a dose-dependent retardation of the m.c. wave frequency. The non-selective beta-adrenoceptor antagonist propranolol (2-200 micrograms/kg, i.a., and 1 mg/kg, i.v.) and the non-selective alpha-adrenoceptor antagonist phentolamine (0.1-1 000 micrograms/kg, i.a.) had no influence on the basal m.c. wave frequency. Propranolol (1-2 mg/kg, i.v.) reduced the effect of isoprenaline and salbutamol (both in the dose range of 0.01-10 micrograms/kg, i.a.) and similarly phentolamine (0.2 and 1 mg/kg, i.a.) reduced the effect of oxymetazoline (0.01-10 microgram/kg, i.a.). It was concluded that during basal conditions in the anesthetized rabbit the m.c. activity functions independently of sympathetic activity and that sympathomimetic agonists acting on beta2-adrenoceptors accelerate the wave frequency, whereas sympathomimetic agonists acting on alpha1 and alpha2-adrenoceptors have a retarding effect, all in a dose-dependent manner.

The influence of lipopolysaccharide (LPS) of Haemophilus influenzae on the ciliary activity of human nasal mucosa was first studied using both a photo-oscillographic apparatus and by bullfrog palate clearance. Neither modification of ciliary activity nor change in bullfrog palate clearance was observed in the early phase after the administration of LPS.

Ciliostasis times of the water-soluble smoke fraction of the University of Kentucky 1R1 Reference Cigarette and experimental cigarettes were compared in the hamster tracheal ring system. Dose-response relations were critically examined, and the assumptions of a dilution bioassay were tested. The model in which a logarithmic transformation was used for both mean time to ciliostasis and smoke sample concentration gave the most satisfactory linear fit. At appropriate concentrations, the ciliostatic responses of the test tobacco smokes were found to be parallel to those of the 1R1, supporting the validity of the dilution bioassay assumptions. Details of experiments and analyses are presented as examples of some of the steps required to establish valid bioassay systems.

The effects of simultaneous exposure to ozone (O3) and sulfuric acid [H2SO4, 0.23 microns volume median diameter (VMD)] and a single exposure to ultrafine (less than 0.1 micron VMD) H2SO4 under various conditions were studied using the infectivity/mortality and the ciliary beating frequency model systems. A 3-h exposure to a combined aerosol of 196 micrograms O3/m3 and 483 or 241 micrograms H2SO4/m3 significantly increased the susceptibility of mice to a laboratory-induced respiratory infection. However, exposure to 543 micrograms ultrafine H2SO4/m3 for 2 h or 365 micrograms/m3 2 h/d for 5 d did not significantly affect this parameter. Upper airway response, as measured by changes in hamster tracheal ciliary beating frequency, was not affected by either a 3-h combined exposure to 196 micrograms O3/m3 and 847 micrograms H2SO4/m3 or a 2-h exposure to 458 micrograms ultrafine H2SO4/m3.

Lysozyme is an important resistance factor in the respiratory defence mechanism. Few reports exist concerning its effects on the ciliary activity of the human nasal mucosa. To clarify the biological activity of lysozyme in ciliary movement, a quantitative study was undertaken using a photo-electrical method to measure ciliary beats in vitro under conditions of constant temperature. Egg-white lysozyme in a 0.01 M sodium phosphate buffer solution, pH 7.4, dose-dependently accelerated the ciliary beats in the human nasal mucosa, removing overlying mucus continuously for at least 60 min. The buffer alone, however, failed to affect ciliary beats. These results indicate that egg-white lysozyme directly promotes the ciliary beats in the human nasal mucosa.

Nasal mucociliary function was studied in 21 healthy subjects before, during and after penicillin administration. No change in the mucociliary function occurred during penicillin treatment. Nor was any difference observed between men and women or between smokers and non-smokers. On the other hand, the measurements revealed a significantly smaller intra-individual than inter-individual variation. As the epithelia of nasal cavity and Eustachian tube are morphologically of the same type it is concluded that the increased incidence of serous otitis media which has been observed after the introduction of penicillin in the treatment of acute otitis media can scarcely be due to an inhibitory effect of penicillin on the mucociliary function of the Eustachian tube.

Exposure to oil mist used in metal work sometimes gives symptoms from skin and airways. This study was performed to evaluate histological and functional respiratory tract disorders. Six male lathe workers aged 31-64 years exposed to oil mist for 4-29 years were examined and compared with matched controls. The investigation included case history, ENT examination, nasal mucociliary function, routine blood tests, IgE, RAST, X-ray of sinus and lungs and biopsy of the nasal mucosa. The mucociliary test showed no difference between the groups. However, all 6 exposed workers had pathological histology findings in the nasal mucosa including lack of cilia, basal cell hyperplasia, goblet cell hyperplasia, squamous metaplasia and subepithelial hyalinization. The biopsies from the controls were mainly normal. The remainder of the investigations revealed no pathology. The study shows that exposure to oil mist--even below the permitted threshold limit--may cause airway symptoms and histological signs comparable to a premature ageing.

The relation of ciliary activity to the partial pressure of carbon dioxide was studied with a photoelectric method on rabbit sinus mucosa in vitro. A dose-response relationship was found, where by ciliary activity was impaired at pCO2 above 5 kPa. The effect of elevated partial pressure of carbon dioxide on ciliary activity in secretory otitis media and sinusitis is discussed.

This study examined the effects of ozone (O3) inhalation on mucociliary clearance from the tracheobronchial tree. Rabbits were exposed for 2 h at 0.1, 0.25, and 0.6 ppm or for 2 h/d for 14 consecutive days at 0.25 and 0.6 ppm. Clearance was assessed by measuring the retention of radioactivity tagged, inert tracer particles inhaled immediately after the single 2-h exposure, or 24 h after 2, 7, or 14 of the daily exposures. Single exposures resulted in a concentration-related trend toward retarded particle clearance, with a statistically significant difference occurring after exposure to 0.6 ppm. Intermittent exposures produced no significant change in clearance rate, although there was a suggestion of retarded clearance at early time points at both 0.25 and 0.6 ppm.

Trachea from albino mice were cut transversely into nearly identical rings and incubated in medium 199 with Hanks salts and HEPES buffer at 37 degrees C. Sodium selenite at 0.5-5 mM depressed the ciliary activity. With 1 and 5 mM sodium selenite, a 50% reduction in the activity index was observed after approximately 5 and 1.5 h, respectively. The ATP content in trachea rings was reduced with 0.05-5 mM sodium selenite, and increasing concentrations gave decreasing amount of ATP after incubation for 4 and 21 h. The rate of protein synthesis as determined by incorporation of radioactive leucine was reduced with 0.5 and 2 mM sodium selenite. The synthesis was reduced quickly by 2 mM sodium selenite, which gave a 30% reduction after incubation for 1 h. It seems that the ATP levels may be used as the most sensitive indication of sodium selenite toxicity in mouse trachea.

A series of experimental designs aimed at collecting and analyzing ciliostasis data in the most efficient and statistically valid manner was examined. The design that proved best for assessing water-soluble tobacco smoke samples in the hamster tracheal ring system utilized two dose levels of the University of Kentucky 1R1 reference cigarette (which functions as a reference as well as a continuing check on the dose-response relation in each experiment) plus several test tobacco smoke samples, each at a single dose level. A pair of animals formed the basis of a randomized block design, with one of the technician X sample preparation degrees of freedom confounded with animal differences within a pair. Assessment of the test samples can thus be expressed in terms of potency relative to the 1R1 reference. Control chart procedures were an essential facet of the system. Details of experiments and methods of data analysis are presented as examples of some of the steps required ot establish valid bioassay systems.

In the present study, we have examined the hitherto unknown effects of long-term exposure to lipopolysaccharide (LPS), possessing the major parts of biological activity of endotoxin, on ciliary activity in the middle ear. Our results show that LPS can affect the ciliary activity in a dose-response fashion: 1) LPS does not impair the ciliary activity up to 168 h if the concentration is 1 ng/ml or less; 2) 10 ng/ml of LPS does not impair ciliary activity in the middle ear close to the tympanic orifice up to 168 h, but can cause reduced activity distal to the orifice after extended exposure (more than 96 h); 3) 100 ng/ml or more of LPS can cause dysfunction of cilia in the tympanic cavity, and in particular 1 microgram/ml or more of LPS can quickly disable the ciliary activity in the middle ear distal to the orifice.

The effect on the mucociliary (m.c.) activity of the parasympathomimetic drug methacholine and its antagonist atropine was studied with the aid of a newly developed animal test model, designed for evaluating the effect of pharmacological substances on the m.c. wave frequency. Methacholine i.a. (0.01-2 micrograms/kg) was found to give a dose-dependent acceleration of the m.c. wave frequency, and the threshold dose 0.03 +/- 0.02 (+/- S.D.) micrograms/kg is suggested to be within physiological limits. The anticholinergic drug atropine i.a. (0.05-0.5 mg/kg) did not influence the basal m.c. wave frequency, but a dose of 0.2 mg/kg reduced or abolished the responses to methacholine (0.05-2 micrograms/kg). These results indicate that the basal m.c. activity functions independently of parasympathetic activity in anesthetized animals, that parasympathomimetic drugs influence the m.c. activity and that the effects of these drugs can be reduced or blocked by atropine.

The effect of whole cigarette smoke on mucociliary (m.c.) activity was recorded in the maxillary sinus in anesthetized rabbits. Puffs of cigarette smoke were delivered in a way that imitates the normal smoking habit in man. Cigarette smoke from nicotinic cigarettes was found to accelerate the m.c. wave frequency in proportion to the nicotine content. Smoke from nicotine-free cigarettes and exposure to oxygen did not influence the m.c. activity. The stimulating effect of smoke drawn from nicotine cigarettes was inhibited by hexamethonium (0.2 mg/kg i.a.) and by atropine (0.2 mg/kg i.a.). It is concluded that the accelerating effect on the m.c. activity of short-term exposure to cigarette smoke might be explained by stimulation of ganglion nicotinic receptors situated on parasympathetic postganglionic nerve cells.

The biological activity of the volatile part of the particulate phase of cigarette-smoke condensate, the semivolatile fraction, has been examined, since the constituents of this material are accessible to selective filtration. Such a process offers a possibility to reduce the biological activity of total cigarette smoke without appreciably affecting the taste. Cigarette-smoke condensate, obtained from domestic American blend type cigarettes, was therefore separated into a nonvolatile and a semivolatile fraction, and the latter was fractionated by liquid-liquid extractions into four subfractions; acids, phenols, bases, and neutrals. The biological activity of these fractions was investigated using six in vitro short-term tests, of which two, the Ames test and the induction of sister chromatid exchanges, provided information on their genotoxicity, and the other four provided information on their cytotoxicity by measuring inhibition of cell growth, inhibition of oxidative metabolism, membrane damage, and ciliotoxicity. Sister chromatid exchanges were found to be induced by the total condensate, the nonvolatile and the semivolatile fractions, and the subfractions derived from the semivolatile fraction, except the bases. The Ames test showed the total condensate and the nonvolatile fraction to contain direct-acting base-pair mutagens as well as indirect-acting frameshift mutagens. While the semivolatile fraction was found nonmutagenic, two of its subfractions, acids and phenols, were shown to contain base-pair mutagens, which did not require metabolic activation. The total condensate and the nonvolatile and semivolatile fractions showed similar activity in the four cytotoxicity tests. Of the semivolatile subfractions, the acids and the phenols exhibited the highest activity and the bases the lowest; the toxicity observed for the neutrals varied with the test system used.

Rabbits were exposed to submicometer sulfuric acid mist (H2SO4) for 1 h/d, 5 d/w for 4 wk, during which time mucociliary clearance was monitored by external in vivo measurements of tagged tracer aerosol retention. One group was exposed orally to 250 micrograms/m3, another to the same concentration via the nose, and a third to 500 micrograms/m3 also via nasal breathing. Clearance was accelerated on specific individual days during the course of the acid exposures, especially at 500 micrograms/m3. In all series, clearance was significantly faster, compared to preexposure controls, during a 2-wk follow-up period after acid exposures had ceased. At the end of this period, the rabbits were sacrificed, and histological sections were obtained from the tracheobronchial tree. Significantly increased epithelial thickness of small conducting airways, compared to sham exposure controls, occurred in rabbits exposed orally at 250 micrograms/m3 or nasally at 500 micrograms/m3, and additionally the lumen of the smallest airways of the former group was narrower than control. The number of airways containing epithelial secretory cells was also significantly greater in these acid exposure groups compared to sham controls. The only change in the rabbits exposed nasally at 250 micrograms/m3 was a significant increase in the number of airways with epithelial secretory cells in the smallest airway classification. The histological alterations provide a basis for observed changes in clearance, and are similar to those found in chronic bronchitis in humans and experimental animals. Differences in site and degree of histological response and degree of physiological change between the two groups exposed to identical acid concentrations appear to have been due to differences in exposure mode, with resultant effects on breathing pattern, aerosol size distribution, and concentration penetrating beyond the upper respiratory tract to specific lung sites.

A test model for in vivo studies of the effect of pharmacological substances on mucociliary activity in the rabbit maxillary sinus is presented. The method permits administration of pharmacological substances and recording of the mucociliary activity under conditions that mimic the physiological situation very closely. The test substances are given intra-arterially. Using a photoelectric technique the mucociliary activity is recorded from a small and limited area of the mucous membrane. There is minimal exposure to external stimuli since normal sinus ventilation is retained. Various factors which may influence the results, e.g. the anesthesia, movements from cardiac and respiratory activity, and the volumes and injection velocities of test doses, are analysed and discussed. When the animal model was tested with serotonin, ATP, and certain cholinergic and adrenergic agents, it was found to be reliable. It permitted easy detection of changes in the mucociliary activity and gave reproducible results.

The in vivo effect of bradykinin on mucociliary (m.c.) activity in the rabbit maxillary sinus was investigated by administering the substance (0.01-10.0 micrograms/kg) via arteria maxillaris and recording the responses with a non-invasive photoelectric technique. Bradykinin accelerated the m.c. activity in a dose-dependent manner in the dose range 0.05-10.0 micrograms/kg. While the action of bradykinin was resistant to pretreatment with indomethacin 10.0 mg/kg, it was considerably weakened by the substance P antagonist [D-Pro2, D-Trp7,9]SP 1 mg/kg. The initial effect of bradykinin was also suppressed by atropine, suggesting that bradykinin accelerates m.c. activity by a dual mechanism comprising both SP and acetylcholine. Bradykinin probably stimulates a reflex arc in the airways involving afferent SP neurons and efferent post-ganglionic parasympathetic neurons.

The influence of sympathetic and parasympathetic controls on regional particle deposition and mucociliary clearance rates was studied in the donkey in vivo. The deposition and clearance characteristics for gamma-tagged monodisperse ferric oxide microspheres were determined for inhalation tests after atropine and methacholine injections and were compared with the characteristics determined for control tests with the same animals and aerosols. Additional tests were performed in which methacholine was injected immediately after the test aerosol inhalation and 2 1/2 h before the particle inhalation. Particle deposition is shifted distally by atropine. Methacholine produces a proximal shift initially and a distal shift 2 1/2 h later. Atropine slows mucociliary transport. Methacholine produces an acceleration in bronchial clearance initially but causes a reduced rate of clearance 2 1/2 h later. Atropine slows mucociliary transport. Methacholine produces an acceleration in bronchial clearance initially but causes a reduced rate of clearance 2 1/2 h later. It also produces an initial surge in tracheal transport, which frequently leads to tracheal mucus refluxing and thereby an increase in average tracheal residence time. The drugs were used to modify the normal deposition and clearance characteristics of a particular animal so that they resembled the normal characteristics of a different animal, suggesting that much of the large intersubject variability may be attributable to different levels of autonomic tone.