The distribution of cationized ferritin receptors on ciliated epithelial cells of rat trachea

The interaction of tracheal cilia with the biphasic mucus layer covering the surface of the mammalian respiratory tract may be influenced by many cell surface coat components including those having an overall negative charge. In order to assess the distribution of ciliary anionic sites, cationized ferritin (CF) was used to label the surface of rat tracheal epithelium. If pieces of trachea were fixed with 3% glutaraldehyde and treated with CF at low (L) (0.08 mg/ml), medium (M) (0.32 mg/ml PBS), or high (H) (0.64 mg/ml PBS) concentrations, the label was distributed evenly over the entire external surface of the ciliary membrane at all concentrations. Unfixed tracheal tissue was also treated with L, M, and H CF for 1 or 5 min at 4 degrees C in order to minimize lateral redistribution of CF receptors. To ensure accessibility of the cell surface to CF the samples were agitated thoroughly during exposure. Exposure for 1 min to L, M, and H CF resulted in a light binding of ferritin particles on all portions of the ciliary membrane with occasional areas of multilayered binding distributed randomly on the ciliary shaft. When unfixed trachea was treated with CF for 5 min at 4 degrees C, CF binding was similar except heavier and more uniform. In no instance was there any preferential binding of CF to the ciliary tips at any of the concentrations used. Moreover, as indicated by the CF binding pattern at L concentrations, high density negative charges are present over almost the entire surface of the cilium. These results suggest that, unlike the ciliary membrane of other organs such as oviduct, negatively charged cell surface coat molecules are present on all areas of the ciliary membrane of rat tracheal epithelia.     

Ultrastructural localization of microtubule-associated proteins (MAP) 1 in cilia of the respiratory tract

The presence and localization of high molecular weight microtubule-associated proteins of the MAP 1 class in ciliated cells of porcine and rat respiratory tract was studied by immunoblotting and immunoelectron microscopy. Ciliary shafts of the porcine tracheal epithelium were isolated using a method that minimizes contamination of the preparation by other cellular fragments and fat. Immunoblotting with rabbit antibodies to bulk MAP 1 from hog brain clearly revealed the presence of anti-MAP 1-immunoreactive high molecular weight proteins of the MAP 1 size in these preparations. To localize MAP 1 proteins at the ultrastructural level, rat and porcine tracheal epithelia were embedded in LR White and subjected to immunogold electron microscopy. Anti-MAP 1-immunoreactive material was found at ciliary shafts and basal bodies, but not at basal feet or ciliary rootlets. Interestingly, the necklace region between the shaft and the basal body of the cilium was hardly reactive with anti-MAP 1 antibodies. This may indicate a reduced stability of ciliary microtubules in this region and could be an explanation why ciliary shafts in general break more easily there than elsewhere.     

Temperature effect on the ciliary beat frequency of human nasal and tracheal ciliated cells.

Even though all human respiratory cilia are similar in structure, they experience a wide range of temperatures between the initial part of the nasal fossae which behave as heat exchangers and the inferior part of the trachea, particularly when we inhale exceedingly cold or hot air. The ciliary beat frequency of ciliated cells from human nasal mucosa and from bronchial mucosa averages 8 Hz when measured at room temperature. In the present study we compared the ciliary beat frequency of human cells from nasal and tracheal mucosa brushings at different temperatures from 5 degrees C to 50 degrees C using two different techniques, ex vivo and in vitro: ex vivo in culture medium less than 24 h after sampling and in vitro after demembranation and reactivation according to a standard procedure developed in our laboratory. Measuring the ATP-reactivated ciliary beat frequency allowed us to check the thermal parameters of the dynein ATPase and all the axonemal machinery. No significant difference in frequency was observed between nasal fossae cilia and tracheal cilia when comparing extreme temperatures in both experimental procedures.     

Respiratory Mucosal Proteome Quantification in Human Influenza Infections

Respiratory influenza virus infections represent a serious threat to human health. Underlying medical conditions and genetic make-up predispose some influenza patients to more severe forms of disease. To date, only a few studies have been performed in patients to correlate a selected group of cytokines and chemokines with influenza infection. Therefore, we evaluated the potential of a novel multiplex micro-proteomics technology, SOMAscan, to quantify proteins in the respiratory mucosa of influenza A and B infected individuals. The analysis included but was not limited to quantification of cytokines and chemokines detected in previous studies. SOMAscan quantified more than 1,000 secreted proteins in small nasal wash volumes from infected and healthy individuals. Our results illustrate the utility of micro-proteomic technology for analysis of proteins in small volumes of respiratory mucosal samples. Furthermore, when we compared nasal wash samples from influenza-infected patients with viral load ≥ 2⁸ and increased IL-6 and CXCL10 to healthy controls, we identified 162 differentially-expressed proteins between the two groups. This number greatly exceeds the number of DEPs identified in previous studies in human influenza patients. Most of the identified proteins were associated with the host immune response to infection, and changes in protein levels of 151 of the DEPs were significantly correlated with viral load. Most important, SOMAscan identified differentially expressed proteins heretofore not associated with respiratory influenza infection in humans. Our study is the first report for the use of SOMAscan to screen nasal secretions. It establishes a precedent for micro-proteomic quantification of proteins that reflect ongoing response to respiratory infection.     

Enzyme-ultracytochemical study of adenylate and guanylate cyclases in normal and pathologic human nasal mucosa

The ultracytochemical localization of adenylate cyclase (AC) and guanylate cyclase B (GC-B) and C (GC-C) activity was studied after stimulation with pituitary adenylate cyclase activating peptide, C-type natriuretic peptide and guanylin, respectively, in normal human respiratory nasal mucosa and mucosa of nasal polyps. To demonstrate these enzymatic activities, we employed enzyme-ultracytochemical methods for electron microscopy. Both normal and pathologic nasal mucosa contained AC, GC-B and GC-C activity. In the upper portion of respiratory epithelium, the enzymes were detected on ciliary and microvillar membranes. In ciliary membranes, GC-B was the predominant form expressed. In goblet cells and in glands of the lamina propria, enzymatic activities were localized mainly on plasma membranes and on membranes lining secretory granules. The results did not reveal any evident differences between the enzymatic activities in normal and pathological nasal mucosa and suggest complementary activities for these enzymes and their stimulators in the regulation of mucociliary transport and glandular secretion.     

Ultrastructural morphology and staining characteristics of Pneumocystis carinii in situ and from bronchoalveolar lavage

The ultrastructure of Pneumocystis carinii obtained from rats by bronchoalveolar lavage (BAL) was compared with organisms in situ. All developmental forms of the organism as seen in situ were present in the lavage fluid. Trophozoites in situ were adhered to type I epithelium, had smooth surfaces, and were interdigitated with the underlying epithelium. Nonadherent trophozoites in situ and trophozoites in lavage fluid were more pleomorphic and irregular in shape with tubular projections extending from all surfaces. Microtubular and nuclear details not reported elsewhere were observed. To enhance the ultrastructural detail of P. carinii obtained by lavage, phosphotungstic and tannic acid fixation, uranyl acetate en bloc staining, and acid phosphatase staining were performed. These techniques enhanced the visibility of membranes, mitochondria, nuclei, and vacuoles. With tannic acid, increased contrast of the organism's cell coat was obtained and differences in staining intensity and thickness related to developmental stages were observed. In lavage samples with few pneumocystis organisms or those specimens heavily contaminated with macrophages, erythrocytes, or other cellular debris, tannic acid allows for easier recognition as other lung materials do not show the same distinctive staining reaction. Lung sections observed after BAL showed intact but damaged epithelial surfaces devoid of organisms. No intracellular organisms were observed. BAL removes organisms from the alveolar lumen as well as adhered organisms and is a useful method for concentrating the various morphologic forms of P. carinii.     

Mechanical damage to rabbit tracheal epithelium from inhaling inert pyrite dust of needle-like structure. Part II.

The ultrastructure of rabbit tracheal epithelium was studied 2, 8 and 26 hours after termination of 8-hour inhalation of ground pyrite dust. Pyrite particles persist in the cytoplasm of ciliated cells throughout the interval followed up. Only through degeneration and elimination of these cells from the epithelium is the inhaled dust gradually removed from the mucous membrane. Goblet cells discharge pyrite-containing mucus into the ciliary border region for a period of 26 hours after cessation of inhalation thus inducing further damage to the ciliary border and the apical regions of the cells even after termination of inhalation. The morphological appearance of the ciliary border, especially in the first phase after termination of inhalation, suggests a disturbance of the self-cleaning function of the eipithelium of respiratory passages. The inhalation of aerosol of needle-like structure causes mechanical damage to tracheal epithelium. Substantial retardation of the onset of epithelial regeneration and slow rate of elimination of the inhaled aerosol from the tracheal mucosa was noted.     

Effect of Pseudomonas aeruginosa rhamnolipids on mucociliary transport and ciliary beating.

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)     

Intercellular spaces between macula densa cells: An ultrastructural study comparing high pressure perfusion fixation with in situ drip-fixation of rat kidney

Summary Polyclonal antibodies have been raised against purified bovine pyrazine-binding protein, a protein that binds the odorant 2-isobutyl-3-methoxypyrazine. These antibodies have been utilized in immunocytochemical experiments to localize the pyrazine-binding protein in bovine nasal mucosa. Tissue fragments, macroscopically identified as olfactory and respiratory mucosa, were fixed in Bouin's fluid and embedded in paraffin. Consecutive serial sections were processed for immunofluorescence studies and restained either with haematoxylin-eosin or with periodic acid Schiff-Alcian Blue. In both olfactory and respiratory mucosa, only seromucous tubulo-acinar glands were specifically labelled. These glands are located in the lamina propria underlying typical respiratory epithelium, even in those tissues that are macroscopically defined as olfactory mucosa.     

280例呼吸道感染儿童呼吸道病毒病原学检出情况及流行规律分析

目的:了解呼吸道感染儿童呼吸道病毒病原学检出情况及其流行规律,为儿童呼吸道感染的预防、诊断及治疗提供病原学依据。方法:选取2016年1月-2017年12月期间中国人民解放军中部战区总医院收治的280例呼吸道感染患儿为研究对象,分析患儿呼吸道分泌物中呼吸道病毒的检出情况,并分析呼吸道感染儿童呼吸道病毒感染与年龄、季节、疾病类型的关系。结果:280例呼吸道感染患儿中共检出98份阳性标本,阳性率为35.00%,其中有2份标本中检出2种病毒感染,混合感染阳性率为0.71%;在所有病毒类型中,呼吸道合胞病毒(RSV)病毒感染阳性率最高。1岁患儿的病毒感染阳性率最高,与其他年龄段病毒感染阳性率比较差异有统计学意义(P0.05)。呼吸道感染患儿春季、冬季的病毒感染阳性率明显高于夏季、秋季(P0.05)。不同呼吸道感染疾病类型患儿病毒感染阳性率比较差异有统计学意义(P0.05),以喘息性肺炎、毛细支气管炎、肺炎患儿病毒感染阳性率较高。结论:RSV是呼吸道感染儿童呼吸道病毒感染的主要致病病原体,1岁的婴幼儿较易感染,春季、冬季为其高发季节,且以肺炎、毛细支气管炎、喘息性肺炎患儿的病毒感染阳性率较高。     

Dual effect of tannic acid on the preservation and ultrastructure of phosphatidyl choline vesicles

Summary The use of tannic acid has been proposed to improve the preservation of phospholipids in tissues. We investigated the effects of tannic acid on the preservation of small unilamellar vesicles, prepared from sonicated aqueous suspensions of phospholipids.With cryo-electron microscopy it is demonstrated that small unilamellar vesicles are formed after sonication of the phospholipid suspensions. Fixation of vesicles without tannic acid results in extraction of the phospholipids during dehydration and embedding. Fixation of vesicles containing phosphatidyl choline with tannic acid, with or without glutaraldehyde, results in a fast (within a second) aggregation of the vesicles and the resulting sediment can be dehydrated and embedded when a postfixation in osmium tetroxide is carried out. Small unilamellar vesicles fixed in this way are retrieved in thin sections as multilamellar vesicles with a periodicity of about 5 nm for dimyristoylphosphatidyl choline and about 6 nm for dioleoylphosphatidyl choline.By using ¹³C-phosphatidyl choline it was also demonstrated that tannic acid prevents to a large extend the extraction of phosphatidyl choline during fixation, dehydration and embedding. This dual effect of tannic acid on phosphatidyl choline, aggregation and fixation, should be considered when using tannic acid in tissue preparation.     

Effects of local anaesthetics (lidocaine) on the structure and function of ciliated respiratory epithelial cells

Summary— Sampling for nasal or bronchial ciliated cells requires the use of anaesthetic agents, but such drugs may interfere with the morphological or functional results. Lidocaine is the most frequently used local anaesthetic. In order to study the morphological and functional effects of lidocaine hydrochloride, we designed an experimental study on ciliated cells from guinea pig and bovine trachea. On guinea pig tracheal specimens, different lidocaine concentrations (0.05, 0.25 and 1%) were tested. Tracheal rings were immersed in either culture medium alone (control) or in different lidocaine concentrations. Measurements of ciliary beat frequency (CBF) were performed by the stroboscopic method. Tracheal rings were consecutively incubated in culture medium alone and a second set of measurements was performed. Tracheal rings were studied by light microscopy after incubation in either 1% lidocaine or in culture medium alone. On bovine tracheal specimens, a coton wool swab impregnated with different lidocaine concentrations (0, 0.25, 1, 2.5 and 5%) was placed in contact with the tracheal mucosa. Three different kinds of samples were collected: the first one was used to study CBF, the second one (0.1 and 5%) was studied by scanning electron microscope (SEM) and the third (0.1 and 5%) by transmission electron microscopy (TEM). The results on guinea pig specimens show a significant but reversible CBF diminution for concentrations of 0.25 and 1% lidocaine and cellular lesions for the concentration of 1%. On bovine specimens a diminution in CBF for concentrations of 2.5 and 5% lidocaine was shown and the SEM study demonstrated obvious lesions on the epithelial surface treated with the 5% concentration. The TEM study showed morphological alterations on respiratory epithelium (deciliated areas, cytoplasmic vacuoles and mitochondrial swelling) for 5% lidocaine concentration. However the axonemal structure of cilia was normal for control and 5% concentration. We concluded that in vitro lidocaine can inhibit the CBF and that high concentrations of lidocaine can damage the respiratory epithelium but without modifications of the axonemal ultrastructure.     

Tannic acid as a stabilizer of liposomal lipids in electron microscopic research

Using electron microscopy the action of tannic acid on the morphology of positively charged liposomes were studied in suspension and after adsorption of the surface membrane of human red blood cell in vitro. After treatment with tannic acid and glutaraldehyde followed by postosmication, an irreversible reconstruction of liposome lipid material with formation of many layers with tight packed lamellae was shown. Similar structures were found on the surface of red blood cells. Monolayer microvesicles 30 nm in diameter were seen after glutaraldehyde fixation postosmication, and final treatment with tannic acid. Similar microvesicles were seen adsorbed on the plasma membrane.     

The use of glutaraldehyde and tannic acid to preserve reconstituted collagen for electron microscopy

The effects of glutaraldehyde and tannic acid on the axial periodicity of collagen have measured. Both fixatives produce axial shrinkage of the collagen but whereas glutaraldehyde produces 7% shrinkage, tannic acid produces only 2% shrinkage. The technique of carbon/platinum shadowing was used to estimate the extent to which the collagen fibrils flatten down when they are dried onto grids for electron microscopy without prior embedding and sectioning. The influence of fixation was studied and it was found that minimum distortion occurred when both tannic acid and glutaraldehyde were used to preserve the protein structure.     

The use of glutaraldehyde and tannic acid to preserve reconstituted collagen for electron microscopy

Summary The effects of glutaraldehyde and tannic acid on the axial periodicity of collagen have been measured. Both fixatives produce axial shrinkage of the collagen but whereas glutaraldehyde produces 7% shrinkage, tannic acid produces only 2% shrinkage. The technique of carbon/platinum shadowing was used to estimate the extent to which the collagen fibrils flatten down when they are dried onto grids for electron microscopy without prior embedding and sectioning. The influence of fixation was studied and it was found that minimum distortion occurred when both tannic acid and glutaraldehyde were used to preserve the protein structure.     

An improved immunocytochemical method for subcellular localization of serotonin in rat enterochromaffin cells

Serotonin-like immunoreactivity (5-HT-LI) has been localized at the ultrastructural level in enterochromaffin (EC) cells of rat gastrointestinal tract. Ultra-thin sections of tissues embedded in epoxy resin were incubated with 5-HT antisera and antibody binding sites were visualized with protein A-gold. Three different antisera were compared and were shown to require different fixation regimens for optimal preservation of 5-HT-LI. For one antiserum, tissues fixed in glutaraldehyde and osmium tetroxide could be used to demonstrate 5-HT-LI in EC cells. Immunocytochemical localization of 5-HT can thus be performed with good ultrastructural preservation of tissues. Quantitative evaluation of the intracellular distribution of 5-HT-LI was performed on EC cells from antrum, duodenum, and proximal colon, fixed in glutaraldehyde only. In all three locations, the majority of the gold particles (90%) in EC cells were localized over the dense core of the secretory granules, while a minor fraction (10%) were localized in parts of the cytoplasm devoid of granules. In EC cells fixed in glutaraldehyde and post-fixed in osmium tetroxide, 5-HT-LI was reduced by about 85%, although intracellular distribution was essentially the same as in cells fixed in glutaraldehyde alone. The results indicate that 5-HT in EC cells is stored mainly in secretory granules, with a small fraction of 5-HT being localized outside the granules.     

Infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent

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.     

Regulation of ciliary beat frequency by the nitric oxide signaling pathway in mouse nasal and tracheal epithelial cells

Objectives

Our purpose was to investigate the role of the nitric oxide (NO) signaling pathway in the regulation of ciliary beat frequency (CBF) in mouse nasal and tracheal epithelial cells.

Methods

We studied the effects of the NO donor l-arginine (L-Arg) and specific inhibitors of the NO signaling pathway on CBF of both nasal and tracheal epithelial cells by using high-speed digital microscopy. We also examined eNOS, sGC β, PKG I and acetylated α tubulin expression in native mouse nasal and tracheal epithelium using immunohistochemical methods.

Results

L-Arg significantly increased CBF of cultured nasal and tracheal epithelial cells, and the effects were blocked by pretreatment with N^G-nitro-l-arginine methyl ester (L-NAME), a NOS inhibitor, with LY-83583, a sGC inhibitor, or with KT-5823, a PKG inhibitor. Positive immunostaining for NO signaling molecules including eNOS, sGC β and PKG I was observed in either nasal or tracheal ciliated epithelium.

Conclusion

NO plays a role in regulating CBF of mouse respiratory epithelial cells via a eNOS–NO–sGC β–cGMP–PKG I pathway.     

Fixation of trypanosomatids for electron microscopy with the glutaraldehyde-tannic acid method.

Epimastigotes from Trypanosoma cruzi and promastigotes from Herpetomonas samuelpessoai were fixed with glutaraldehyde-tannic acid. Different concentrations of tannic acid were tested. With this technique the cellular membranes appear in negative contrast offering the same aspect as seen in cells fixed in glutaraldehyde only without post-fixation in osmium tetraoxide. An electron-dense deposit appears on the surface which possibly represents positively charged groups. The structure of the sub-pellicular microtubules appears well defined and it was possible to distinguish the 13 protofilaments which compose the microtubule wall.