Scanning electron microscopy ofMycoplasma pneumoniae on the membrane of individual ciliated tracheal cells

Summary Cell monolayer cultures were prepared from hamster tracheal explants by a collagenase exposure and subsequent incubation in Waymouth’s MAB 87/3 medium. The epithelial outgrowth occurred on glass cover slips. Cilia on the monolayers continued to beat normally after the “parent” explant was removed. Monolayer cultures infected withMycoplasma pneumoniae had significant amounts of attachment. A morphological analysis of the attachment was conducted with scanning electron microscopy. Clusters, cocci, and filaments ofM. pneumoniae all attached to the epithelial cells, but the filaments were especially common. Mycoplasmas were seen in association with both ciliated and nonciliated cell membranes. On ciliated cells, mycoplasmas were on the ciliary strands and on the cell membrane. When located immediately adjacent to or in between cilia, mycoplasmas were oriented vertically with the constricted attachment tip oriented down toward the host cell membrane. When located more than a micron away from the ciliary fibers, mycoplasmas lay horizontally along the epithelial cell membrane. The photographic data suggest that clusters or “sperules” of mycoplasmas may liberate individual mycoplasmas that attach to the cell membrane. It appears that the receptor sites forM. pneumoniae are rather uniformly distributed along the ciliated cell membrane, and are not restricted to the interciliary areas. Electron microscopy was done with the cooperation of Dr. R. Macleod and the staff of the Center for Electron Microscopy at the University of Illinois. Critical editorial review was provided by C. Dayton. This investigation was supported in part by grants to M. G. G. from the National Institute of Allergy and Infectious Diseases (AI 12559) and the National Heart, Lung, and Blood Institute (HL 23806), Bethesda, Maryland.     

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

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

Adsorption of Mycoplasma pneumoniae to Neuraminic Acid Receptors of Various Cells and Possible Role in Virulence

Monkey, rat, and chicken tracheal epithelial cells, as well as monkey, rat, guinea pig, and chicken erythrocytes, adsorbed firmly to colonies of Mycoplasma pneumoniae and M. gallisepticum. Colonies of M. pulmonis also adsorbed erythrocytes but with less avidity than M. pneumoniae or M. gallisepticum; unlike the latter organisms, M. pulmonis did not adsorb tracheal epithelial cells. Colonies of M. orale type 1 and M. orale type 3 adsorbed only chicken red cells. Other mycoplasma species tested, including four of human origin and one of animal origin, did not adsorb red cells or epithelial cells. M. pneumoniae and M. gallisepticum appeared to attach to erythrocytes or tracheal epithelial cells by neuraminic acid receptors on these cells, whereas M. orale types 1 and 3 and M. pulmonis seemed to utilize another type or other types of receptors. Pretreatment of red cells or tracheal epithelial cells with receptor-destroying enzyme, neuraminidase, or influenza B virus removed the adsorption receptors for M. pneumoniae. Similarly, pretreatment of M. pneumoniae colonies with neuraminic acid-containing materials prevented adsorption of erythrocytes or respiratory tract cells. The adsorption sites on M. pneumoniae were specifically blocked by homologous but not heterologous antisera. This property made it possible to study the nature of the mycoplasma adsorption sites by testing the capacity of different fractions of the organism to block the action of adsorption-inhibiting antibodies. Such studies suggested that the mycoplasma binding sites were probably lipid or lipoprotein in nature. The glycerophospholipid hapten was implicated as one such site, since this serologically active hapten blocked the action of hemadsorption-inhibiting antibodies in M. pneumoniae rabbit antiserum. The affinity of M. pneumoniae for respiratory tract epithelium, unique among the mycoplasmas that infect man, may play a role in virulence, since this type of attachment provides an unusual opportunity for peroxide, secreted by the organism, to attack the tissue cell membrane without being rapidly destroyed by catalase or peroxidase present in extracellular body fluids.     

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

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

Mucociliary Clearance Defects in a Murine In Vitro Model of Pneumococcal Airway Infection

Mucociliary airway clearance is an innate defense mechanism that protects the lung from harmful effects of inhaled pathogens. In order to escape mechanical clearance, airway pathogens including Streptococcus pneumoniae (pneumococcus) are thought to inactivate mucociliary clearance by mechanisms such as slowing of ciliary beating and lytic damage of epithelial cells. Pore-forming toxins like pneumolysin, may be instrumental in these processes. In a murine in vitro airway infection model using tracheal epithelial cells grown in air-liquid interface cultures, we investigated the functional consequences on the ciliated respiratory epithelium when the first contact with pneumococci is established. High-speed video microscopy and live-cell imaging showed that the apical infection with both wildtype and pneumolysin-deficient pneumococci caused insufficient fluid flow along the epithelial surface and loss of efficient clearance, whereas ciliary beat frequency remained within the normal range. Three-dimensional confocal microscopy demonstrated that pneumococci caused specific morphologic aberrations of two key elements in the F-actin cytoskeleton: the junctional F-actin at the apical cortex of the lateral cell borders and the apical F-actin, localized within the planes of the apical cell sides at the ciliary bases. The lesions affected the columnar shape of the polarized respiratory epithelial cells. In addition, the planar architecture of the entire ciliated respiratory epithelium was irregularly distorted. Our observations indicate that the mechanical supports essential for both effective cilia strokes and stability of the epithelial barrier were weakened. We provide a new model, where - in pneumococcal infection - persistent ciliary beating generates turbulent fluid flow at non-planar distorted epithelial surface areas, which enables pneumococci to resist mechanical cilia-mediated clearance.     

Chibby promotes ciliary vesicle formation and basal body docking during airway cell differentiation

Airway multiciliated epithelial cells play crucial roles in the mucosal defense system, but their differentiation process remains poorly understood. Mice lacking the basal body component Chibby (Cby) exhibit impaired mucociliary transport caused by defective ciliogenesis, resulting in chronic airway infection. In this paper, using primary cultures of mouse tracheal epithelial cells, we show that Cby facilitates basal body docking to the apical cell membrane through proper formation of ciliary vesicles at the distal appendage during the early stages of ciliogenesis. Cby is recruited to the distal appendages of centrioles via physical interaction with the distal appendage protein CEP164. Cby then associates with the membrane trafficking machinery component Rabin8, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rab8, to promote recruitment of Rab8 and efficient assembly of ciliary vesicles. Thus, our study identifies Cby as a key regulator of ciliary vesicle formation and basal body docking during the differentiation of airway ciliated cells.     

Functional activity of ciliated outgrowths from cultured human nasal and tracheal epithelia

Primary cultures of respiratory epithelium were produced as outgrowths from human fetal and adult tracheal and nasal polyp explants. Video recordings of the epithelial cell outgrowths were carried out after 5 days of culture and the ciliary beating frequency was analyzed by using a video technique. Uniform fields of differentiated ciliated cells were observed near the edge of the explant. In the transition region of the outgrowth from the explant to the outgrowth periphery, isolated ciliated cells were present, as well as cells with fused cilia. The ciliary beating frequency of the outgrowth of well-differentiated ciliated cells (13.5 +/- 1.4 Hz) was significantly higher (p less than 0.001) than the beating frequency of both the explant (11.9 +/- 0.7 Hz) and the ciliated cells with fused cilia (9.8 +/- 1.7 Hz). The same differentiation stages and functional activities were observed in the outgrowth cultures, whatever their origin. These in vitro models are comparable with each other and therefore could be useful for studying the ciliogenesis and functional activity of the human respiratory epithelium.     

Characterization of pediatric cystic fibrosis airway epithelial cell cultures at the air-liquid interface obtained by non-invasive nasal cytology brush sampling

Background

In vitro systems of primary cystic fibrosis (CF) airway epithelial cells are an important tool to study molecular and functional features of the native respiratory epithelium. However, undifferentiated CF airway cell cultures grown under submerged conditions do not appropriately represent the physiological situation. A more advanced CF cell culture system based on airway epithelial cells grown at the air-liquid interface (ALI) recapitulates most of the in vivo-like properties but requires the use of invasive sampling methods. In this study, we describe a detailed characterization of fully differentiated primary CF airway epithelial cells obtained by non-invasive nasal brushing of pediatric patients.

Methods

Differentiated cell cultures were evaluated with immunolabelling of markers for ciliated, mucus-secreting and basal cells, and tight junction and CFTR proteins. Epithelial morphology and ultrastructure was examined by histology and transmission electron microscopy. Ciliary beat frequency was investigated by a video-microscopy approach and trans-epithelial electrical resistance was assessed with an epithelial Volt-Ohm meter system. Finally, epithelial permeability was analysed by using a cell layer integrity test and baseline cytokine levels where measured by an enzyme-linked immunosorbent assay.

Results

Pediatric CF nasal cultures grown at the ALI showed a differentiation into a pseudostratified epithelium with a mucociliary phenotype. Also, immunofluorescence analysis revealed the presence of ciliated, mucus-secreting and basal cells and tight junctions. CFTR protein expression was observed in CF (F508del/F508del) and healthy cultures and baseline interleukin (IL)-8 and IL-6 release were similar in control and CF ALI cultures. The ciliary beat frequency was 9.67 Hz and the differentiated pediatric CF epithelium was found to be functionally tight.

Conclusion

In summary, primary pediatric CF nasal epithelial cell cultures grown at the ALI showed full differentiation into ciliated, mucus-producing and basal cells, which adequately reflect the in vivo properties of the human respiratory epithelium.

     

Utilization of Neuraminic Acid Receptors by Mycoplasmas

Erythrocytes and H-HeLa cells were treated with neuraminidase and then compared with untreated cells for their ability to adsorb to mycoplasma colonies or be agglutinated by suspensions of the mycoplasmas. Of the 17 mycoplasma serotypes examined, only 4 were found to use neuraminic acid receptors; these were Mycoplasma pneumoniae, M. gallisepticum, M. synoviae, and mycoplasma WR1. Not all strains of a serotype behaved alike. Thus, removal of receptors on erythrocytes for one strain of M. gallisepticum required at least 100 times the concentration of neuraminidase needed to remove them for another strain. The mechanism of attachment of erythrocytes to mycoplasma colonies does not appear to be the same as that for attachment to mycoplasmas in suspension.     

Organization of actin microfilaments in the apical border of oviduct ciliated cells

Actin microfilaments were localized in quail oviduct ciliated cells using decoration with myosin subfragment S1 and immunogold labeling. These polarized epithelial cells show a well developed cytoskeleton due to the presence of numerous cilia and microvilli at their apical pole. Most S1-decorated microfilaments extend from the microvilli downward towards the upper part of the ciliary striated rootlets with which they are connected. From the microvillous roots, a few microfilaments connect the proximal part of the basal body or the basal foot associated with the basal body. Microfilament polarity is shown by S1 arrowheads pointing away from the microvillous tip to the cell body. Furthermore, short microfilaments are attached to the plasma membrane at the anchoring sites of basal bodies and run along the basal body. The polarity of these short microfilaments is directed from the basal body anchoring fibers downward to the cytoplasm. At the cell periphery, microfilaments from microvillous roots and ciliary apparatus are connected with those of the circumferential actin belt which is associated with the apical zonula adhaerens. Together with the other cytoskeletal elements, the microfilaments increase ciliary anchorage and could be involved in the coordination of ciliary beating. Moreover, microvilli surrounding the cilia probably modify ciliary beating by offering resistance to cilium bending. The presence of microvilli could explain the fact that mainly the upper part of the cilia appanars to be involved in the axonemal bending in metazoan ciliated cells.     

Immunodetection of annexins 1 and 2 in ciliated cells from quail oviduct.

Annexins 1 and 2 are Ca(2+)-binding proteins related to the cytoskeletal proteins which have been reported to bind in a calcium-dependent manner of F-actin and phospholipids in vitro. Proteins immunologically related to the brain 37-kDa annexin 1 and 36-kDa annexin 2 were characterized by immunoblotting epithelial ciliated cells from quail oviduct. They were detected by immunofluorescence in ciliated as well as glandular cells, using antisera and purified antibodies directed against pig brain annexins. The pattern of labeling was found in the apical part of both cell types, with close membrane association. However, a wider distribution was observed in mature ciliated cells: annexins were localized in the well developed cytoskeletal meshwork in which the ciliary apparatus is tightly anchored. After immunogold labeling, annexins 1 and 2 were located in the same area as spectrin 240/235 and at the connection sites of F-actin; both these cytoskeletals proteins were associated with the appendages of the basal body. In contrast, annexins were not detected in immature epithelial cells, while actin and spectrin were present. During ciliogenesis, the staining gradually appeared associated with the lateral and apical membranes. In this cellular model, the annexins may function during exocytosis in gland epithelial cells, where a close cytoskeleton-membrane association is observed; moreover, in ciliated cells, a relationship between cytoskeletal elements of the terminal web and annexins may exist.     

Internalization and intracellular survival of Mycoplasma pneumoniae by non-phagocytic cells

Current theory holds that mycoplasmas remain attached to the surface of epithelial cells although some mycoplasmas have evolved mechanisms for entering host cells that are not naturally phagocytic. The ability of Mycoplasma pneumoniae strain M129 to invade and survive within host cells was studied using a HeLa cell line and a human lung carcinoma cell line (A549). The invasion process into the eukaryotic cells was studied qualitatively by confocal laser scanning microscopy and quantitatively by the gentamicin resistance assay. Internalization was found with A549 cells but not with HeLa cells. Internalization was dependent on the duration of the infection and on temperature. The organism, detected in the cytoplasm and perinuclear regions, survived within the host cells for prolonged periods of time. The intracellular location of M. pneumoniae is obviously a privileged niche, well protected from the immune system and from the action of many antibiotics and may explain the pathogenic potential of this organism.     

Mechanical sensitivity and cell coupling in the ciliated epithelial cells ofMytilus edulis gill

Summary Transmission electron microscopy has not provided strong evidence for gap junctions inMytilus edulis gill tissue, in spite of extensive physiological evidence for coupled ciliary arrest in lateral cells and coupled activation in abfrontal cells. To investigate the kinds and relative distribution of cell junctions and also to determine whether ciliary membrane particle differences exist in these two types of oppositely mechanically sensitive cells, we analyzed the structure of these and two other ciliated cell types (frontal and laterofrontal) by freeze-fracture replication. Gap junctions occur in all four ciliated cell types, but they are relatively small and of variable morphology, often consisting of elongate, winding complexes of membrane particles. Statistically, such structures rarely would be recognized as gap junctions in thin sections. Gap junctions appear to be most abundant between the highly coupled abfrontal cells, minimal between laterofrontal cells, and not evident in the epithelial cells that separate coupled ciliated cell types. The ciliary necklaces of the mechanically activated abfrontal cilia are typically 4- or 5-stranded while those of the remaining three cell types are mainly 3-stranded. In developing gill tips, ciliated cells have abundant gap junctions and newly formed cilia have a full complement of necklace particles. Nascent lateral cilia are not mechanically sensitive, indicating that the acquisition of mechanosensitivity does not correlate with the presence of ciliary necklace or other membrane particles. Lateral and laterofrontal cells become sensitive to neurotransmitters soon after the appearance of the latter during development, but mechanosensitivity of both lateral and abfrontal cells arises substantially later.     

Electron microscopic study of intestinal epithelium of <Emphasis Type="Italic">Saccoglossus mereschkowskii</Emphasis> (Enteropneusta,Hemichordata)

The epithelium of the hepatic region of the intestine in Saccoglossus mereschkowskii, a representative of enteropneusts (Enteropneusta, Hemichordata), a group located at the base of Chordata, has been studied by using electron microscopy. The ultrastructure of ciliated and granular epithelial cells, elements of the intraepithelial nerve layer, and intercellular junctions are characterized. The data on the details of the structure of the ciliary apparatus and the system of ciliary rootlets are presented. Justification is provided for the presence of a complicated construction in the ciliated cells, a supportive carcass of cilia that performs a mechanical stabilizing function, and possibly the synchronization of the ciliary movement. The existence of cilia with two centrioles is considered as adaptation to the high functional load on the ciliary apparatus. Well-developed bundles of myofilaments have been revealed in the cytoplasm of the basal parts of ciliated cells, which characterizes these cells as epitheliomuscular. Peculiarities indicating the role of ciliated cells in absorption are described, as well as the capability of these cells for balloon-like secretion. Data are presented on the accumulation of reserved nutritional substances in the cell cytoplasm in the form of lipids and glycogen. With respect to their function, ciliated cells are determined as the ciliated secretory-absorptive epitheliomuscular cells. The location of secretory granules in both apical and basal parts of granular cells indicates the exocrine-endocrine function of these cells. There are no typical endocrine cells in the intestinal epithelium of S. mereschkowskii. Several types of granules are described in the cytoplasm of nerve fibers. Junctions between nerve fibers and basal parts of ciliated and granular epithelial cells have been revealed; the neural regulation of the contractile and secretory functions of epithelial cells is assumed. The intestinal epithelium of enteropneusts is presumed to contain a regulatory neuroendocrine system composed of receptor cells of the open type, secretory endocrine-like cells, and of nerve elements of the nervous layer.     

Toxic effects of sulfur mustard on respiratory epithelial cells in culture

Sulfur mustard (SM) is known to induce cutaneous injury and to cause acute damage to the respiratory tract. Although skin vesication has been demonstrated on human epidermal keratinocytes in culture, no study has been carried out to analyze the effects of SM on the ultrastructural and functional activity of surface respiratory epithelial cells. To evaluate this SM toxicity, we developed an in vitro model of respiratory epithelial cells in primary culture. The study was performed on surface epithelial cells from rabbit trachea cultured according to the explant-outgrowth technique. The functional activity of the cultures was evaluated by measuring the ciliary beating frequency (CBF) of the ciliated cells with a videomicroscopic method. The morphological aspects of the cells were analyzed by light and electron microscopy. Addition of 0.1 mM SM directly into the culture medium produced a sudden and irreversible CBF inhibition, first observed after 2 hr on the ciliated cells of the outgrowth periphery. The arrest of the ciliary beating progressively reached the whole surface of the outgrowth and was simultaneously observed with a detachment of the outgrowth cells. It began at the outgrowth border, leading to the exfoliation of cell sheets, and then to the whole culture after 48 hr. Morphological damage was expressed by intense vacuolisation and disorganization of cytoplasmic and nuclear structures. These findings suggest that the detachment of the respiratory epithelial cells from the matrix represents a major toxic effect of 0.1 mM SM. SM dramatically affects the viability of respiratory epithelial cells in culture. Moreover, the sudden CBF inhibition is more likely due to the death of the ciliated cells than to a specific ciliotoxic effect of SM.Abbreviations CBF ciliary beating frequency - HEPES N2-hydroxyethylpiperazine-N'2ethanesulfonic acid - PBS phosphate buffer saline - SM sulfur mustard - TEM transmission electron microscopy     

Isolation, cell culture and immunocytochemical characterization of oviduct epithelial cells of the cow

Incubation of cow oviducts flushed with 0.1 mg collagenase/ml, for 90 min helped to dislodge large numbers of ciliated and secretory cells. About 90-95% of the isolated epithelial cells were viable. The epithelial cells suspended in DMEM:F-12 + 10% serum attached to the plastic culture dish in 18-20 h after seeding. The ciliated cells which attached to the plastic dish lost their cilia after 4-5 days in culture. The attached cells, which proliferated to form a confluent monolayer 8-10 days after seeding in a 35-mm dish, could be subcultured at least 3 successive times. Some cell aggregates which did not attach to the culture dish proliferated into floating balls of cells. The ciliated cells in the unattached floating colonies maintained the ciliary movement for 9-10 days in the same culture medium. The primary cultures of the ciliated and the secretory cells maintained most of the histoarchitecture observed in intact epithelium. The secretory cells maintained their secretory activity of specific proteins in culture as indicated by immunocytology. The cultured cells contained keratin, a specific cytoskeletal component of epithelial cells.     

Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology

Gene therapy for cystic fibrosis (CF) lung disease requires efficient gene transfer to airway epithelial cells after intralumenal delivery. Most gene transfer vectors so far tested have not provided the efficiency required. Although human respiratory syncytial virus (RSV), a common respiratory virus, is known to infect the respiratory epithelium, the mechanism of infection and the epithelial cell type targeted by RSV have not been determined. We have utilized human primary airway epithelial cell cultures that generate a well-differentiated pseudostratified mucociliary epithelium to investigate whether RSV infects airway epithelium via the lumenal (apical) surface. A recombinant RSV expressing green fluorescent protein (rgRSV) infected epithelial cell cultures with high gene transfer efficiency when applied to the apical surface but not after basolateral inoculation. Analyses of the cell types infected by RSV revealed that lumenal columnar cells, specifically ciliated epithelial cells, were targeted by RSV and that cultures became susceptible to infection as they differentiated into a ciliated phenotype. In addition to infection of ciliated cells via the apical membrane, RSV was shed exclusively from the apical surface and spread to neighboring ciliated cells by the motion of the cilial beat. Gross histological examination of cultures infected with RSV revealed no evidence of obvious cytopathology, suggesting that RSV infection in the absence of an immune response can be tolerated for >3 months. Therefore, rgRSV efficiently transduced the airway epithelium via the lumenal surface and specifically targeted ciliated airway epithelial cells. Since rgRSV appears to breach the lumenal barriers encountered by other gene transfer vectors in the airway, this virus may be a good candidate for the development of a gene transfer vector for CF lung disease.     

Molecular basis for cytadsorption of Mycoplasma pneumoniae.

Hemadsorbing (HA+) virulent Mycoplasma pneumoniae and spontaneously derived nonhemadsorbing (HA-) avirulent mutants were compared by biochemical and ultrastructural techniques in an attempt to understand the molecular basis for cytadsorption. Lactoperoxidase-catalyzed iodination of intact mycoplasmas indicated that both virulent and avirulent mycoplasmas displayed similar surface protein patterns. A specific external protein, P1 (molecular weight, 165,000), previously implicated as a major ligand mediating attachment, was readily detected in HA+ and HA- mycoplasma strains. However, immunoferritin electron microscopy, with monospecific antibody against P1, revealed that differences in P1 topography existed among these strains. Only virulent mycoplasmas exhibited high concentrations of P1 at the terminal organelle. Avirulent mycoplasmas which possessed P1 showed no P1 clustering at the terminus. Both virulent M. pneumoniae and avirulent P1-containing mutants possessed numerous less dense P1 regions along the mycoplasma surface. Not surprisingly, an HA- mutant lacking P1 exhibited only background immunoferritin labeling. Negative staining of intact mycoplasmas revealed a well-defined, naplike terminus (associated with P1 clusters) confined at the tip of virulent M. pneumoniae. Previous characterization of HA+ virulent and HA- avirulent strains of M. pneumoniae by one- and two-dimensional polyacrylamide gel electrophoresis suggests that identified groups of mycoplasma proteins, lacking in specific HA- mycoplasmas, regulate the physical arrangement of P1 and the ultrastructure of the terminus, thus influencing adherence to the respiratory epithelium and virulence.     

In vitro development and characterization of a manatee bronchial cell line

In the present study, culture conditions that promote the growth and differentiation of manatee respiratory tract epithelial cells toward a mucociliary phenotype were determined. Characterization of a manatee-specific cell line enables investigators to conduct in vitro testing where live-animal experimentation is not possible. Cell cultures were established from both explants and enzymatically dissociated cells that were isolated from manatee bronchial tissue. To modulate their differentiation, bronchial epithelial cells were grown on Transwell collagen membranes either submerged or at an air-liquid interface. Growth on a collagen membrane at an air-liquid interface and medium supplemented with retinoic acid was required to promote a mucociliary phenotype. When cells were grown in submerged cultures without retinoic acid, they appeared more squamous and were not ciliated. Intracellular keratin proteins were detected in both submerged and interface cultures. Cultured manatee bronchial epithelial cells will facilitate future studies to investigate their potential role in pulmonary disease associated with brevetoxicosis after exposure to the red-tide organism, Karenia brevis.