Influence of immunisation with Mycobacterium bovis bacillus Calmette-Guérin on the sensitisation to inhaled allergens after infection with respiratory syncytial virus.

Respiratory syncytial virus (RSV) may play an important role in allergic diathesis by creating a Th2-type immune response. Mycobacterium bovis bacillus Calmette-Guérin (BCG) is known to induce a Th1-type immune response, but the association of BCG vaccination and the suppression of allergy development remain controversial. We investigated the influence of BCG vaccination on the immune response to RSV in a mouse model. Balb/c mice were BCG vaccinated, RSV infected and ovalbumin (OVA) challenged. Mice were sacrificed one, two and four weeks after allergen exposure. Bronchoalveolar lavage was performed. Alveolar macrophages and lymphocytes from spleens and lung-associated lymph nodes were investigated for cytokine production and cell proliferation. Serum was tested for allergen-specific immunoglobulin-E (IgE). Lung eosinophilia was diminished by BCG immunisation. OVA-specific serum IgE was increased regardless of prior BCG vaccination. Interleukin-4 secretion of spleen lymphocytes increased in BCG-vaccinated mice only one week after allergen exposure but was comparable to non-vaccinated mice at four weeks. The reactivity of spleen lymphocytes towards concanavalin-A to secrete interferon-gamma was increased in the vaccinated group at the end of the observation period. Interleukin-6 and tumour necrosis factor-alpha secretion of alveolar macrophages as well as proliferation of stimulated thoracic lymph node cells were increased and prolonged in vaccinated mice. BCG immunisation led to a local suppression of the allergic reaction within the lung. No reduction of systemic IgE production was observed. Further studies are necessary to determine a possible time dependence of BCG immunisation.

Phosphatidylcholine molecular species in lung surfactant: composition in relation to respiratory rate and lung development.

Surfactant reduces surface tension at the air-liquid interface of lung alveoli. While dipalmitoylphosphatidylcholine (PC16:0/ 16:0) is its main component, proteins and other phospholipids contribute to the dynamic properties and homeostasis of alveolar surfactant. Among these components are significant amounts of palmitoylmyristoylphosphatidylcholine (PC16:0/ 14:0) and palmitoylpalmitoleoylphosphatidylcholine (PC16:0/ 16:1), whereas in surfactant from the rigid tubular bird lung, PC16:0/14:0 is absent and PC16:0/16:1 strongly diminished. We therefore hypothesized that the concentrations of PC16:0/14:0 and PC16:0/16:1 in surfactants correlate with differences in the respiratory physiology of mammalian species. In surfactants from newborn and adult mice, rats, and pigs, molar fractions of PC16:0/14:0 and PC16:0/16:1 correlated with respiratory rate. Labeling experiments with [methyl-(3)H]choline in mice and perfused rat lungs demonstrated identical alveolar proportions of total and newly synthesized PC16:0/14:0, PC16:0/16:1, and PC16:0/16:0, which were much higher than those of other phosphatidylcholine species. In surfactant from human term and preterm neonates, fractional concentrations not only of PC16:0/16:0 but also of PC16:0/14:0 and PC16:0/ 16:1 increased with maturation. Our data emphasize that PC16:0/14:0 and PC16:0/16:1 may be important surfactant components in alveolar lungs, and that their concentrations are adapted to respiratory physiology.

Pulmonary surfactant in birds: coping with surface tension in a tubular lung.

As birds have tubular lungs that do not contain alveoli, avian surfactant predominantly functions to maintain airflow in tubes rather than to prevent alveolar collapse. Consequently, we have evaluated structural, biochemical, and functional parameters of avian surfactant as a model for airway surfactant in the mammalian lung. Surfactant was isolated from duck, chicken, and pig lung lavage fluid by differential centrifugation. Electron microscopy revealed a uniform surfactant layer within the air capillaries of the bird lungs, and there was no tubular myelin in purified avian surfactants. Phosphatidylcholine molecular species of the various surfactants were measured by HPLC. Compared with pig surfactant, both bird surfactants were enriched in dipalmitoylphosphatidylcholine, the principle surface tension-lowering agent in surfactant, and depleted in palmitoylmyristoylphosphatidylcholine, the other disaturated phosphatidylcholine of mammalian surfactant. Surfactant protein (SP)-A was determined by immunoblot analysis, and SP-B and SP-C were determined by gel-filtration HPLC. Neither SP-A nor SP-C was detectable in either bird surfactant, but both preparations of surfactant contained SP-B. Surface tension function was determined using both the pulsating bubble surfactometer (PBS) and capillary surfactometer (CS). Under dynamic cycling conditions, where pig surfactant readily reached minimal surface tension values below 5 mN/m, neither avian surfactant reached values below 15 mN/m within 10 pulsations. However, maximal surface tension of avian surfactant was lower than that of porcine surfactant, and all surfactants were equally efficient in the CS. We conclude that a surfactant composed primarily of dipalmitoylphosphatidylcholine and SP-B is adequate to maintain patency of the air capillaries of the bird lung.

Metabolism of surfactant phosphatidylcholine molecular species in cftr(tm1HGU/tm1HGU) mice compared to MF-1 mice.

In cftr(tmIHGU/m1HGU) mice, an animal model designed to study pathophysiologic alterations due to the CFTR defect found in cysticfibrosis, surfactant phospholipids of bronchoalveolar lavage fluid (BALF) are increased. To study the metabolical basis of such increases, we intraperitoneally injected cft(tm1HGU/tm1HGU) mice [methyl-3H]choline and measured [methyl-3H]choline incorporation into phosphatidylcholine (PC) molecular species of lung tissue and BALF after 1.5 to 24 hours. MF1 and MF1 x cftr(tm1HGU/tm1HGU) hybrid mice served as controls. In tissue [methyl-3H]choline incorporation into total PC was constant for 24 hours and identical in control and cftr(tmIHGU/m1HGU) mice. However, from 7.5 to 24 hours there was a shift of [methyl-3H]choline incorporation from palmitoyloleoyl-PC and palmitoyllinoleoyl-PC towards PC species enriched in surfactant, dipalmitoyl-PC, palmitoylmyristoyl-PC, and palmitoylpalmitoleoyl-PC. The relative and absolute 3H-labels of PC species were identical for cftr(tmIHGU/m1HGU) compared to control mice. In BALF [methyl-3H]choline of total PC increased from 1.5 to 24 hours (R2 > .98), mainly due to [methyl-3H]choline-labelled dipalmitoyl-PC, in all experimental groups. In BALF from cftr(tmIHGU/m1HGU) mice, the [methyl-3H]choline label of total PC and individual PC species was significantly increased over control values after 24 hours, but not after 1.5 to 6 hours. Numbers and composition of BALF cells were not different between controls and cftr(tmIHGU/m1HGU) mice. We, conclude that increased alveolar phospholipid in cftr(tmIHGU/m1HGU) mice is likely due to decreased reuptake of surfactant.

Mucosal immunity and viral infections.

The mucosal surfaces are the first portals of entry for most infectious agents, among which respiratory and intestinal viruses are of greatest epidemiological importance. To combat these infections, the immune system uses unspecific and specific mechanisms. Unspecific responses include the production of virus-induced cytokines, such as type 1 interferons and natural killer (NK) cell activity, while specific immune responses mainly depend on cytotoxic T cells, which are important especially in the early course of a viral infection, and on antibodies. At the mucosal sites, antiviral secretory IgA antibodies play a major role in clearing viral infections and preventing or modifying disease after re-exposure. Passive transfer of virus-specific antibodies has been used in experimental and clinical settings to prevent or treat viral mucosal infections. In the future, the development of new mucosal vaccines promises to have the strongest impact on the epidemiology of viral infections.

Pulmonary and gastric surfactants. A comparison of the effect of surface requirements on function and phospholipid composition.

Surfactant is present in the alveoli and conductive airways of mammalian lungs. The presence of surface active agents was, moreover, demonstrated for avian tubular lungs and for the stomach and intestine. As the surface characteristics of these organs differ from each other, their surfactants possess distinct biochemical and functional characteristics. In the stomach so-called 'gastric surfactant' forms a hydrophobic barrier to protect the mucosa against acid back-diffusion. For this purpose gastric mucosal cells secrete unsaturated phosphatidylcholines (PC), but no dipalmitoyl-PC (PC16:0/16:0). By contrast, surfactant from conductive airways, lung alveoli and tubular avian lungs contain PC16:0/16:0 as their main component in similar concentrations. Hence, there is no biochemical relation between gastric and pulmonary surfactant. Alveolar surfactant, being designed for preventing alveolar collapse under the highly dynamic conditions of an oscillating alveolus, easily reaches values of <5 mN/m upon cyclic compression. Surfactants from tubular air-exposed structures, however, like the conductive airways of mammalian lungs and the exclusively tubular avian lung, display inferior compressibility as they only reach minimal surface tension values of approximately 20 mN/m. Hence, the highly dynamic properties of alveolar surfactant do not apply for surfactants designed for air-liquid interfaces of tubular lung structures.

Safety and immunogenicity of an intranasal Pseudomonas aeruginosa hybrid outer membrane protein F-I vaccine in human volunteers.

A hybrid protein [Met-Ala-(His)(6) OprF(190-342)-OprI(21-83)] consisting of the mature outer membrane protein I (OprI) and amino acids 190-342 of OprF of Pseudomonas aeruginosa was expressed in Escherichia coli and purified by Ni(2+) chelate-affinity chromatography. After several studies in healthy volunteers, as well as in patients, had proven the tolerability and immunogenicity of the the OprF-OprI vaccine, after intra-muscular application, we developed an emulgel for intranasal immunization. For this purpose we combined a highly concentrated OprF-I with sodium dodecylsulfate as vehicle and the gel matrix natriumlauryl sulfate. After safety and pyrogenicity evaluations in animals, eight healthy adult human volunteers received the OprF-I gel intranasally three times at 2-week intervals. The vaccination was well tolerated and no side effects were observed. An antibody induction (IgG and IgA) could be detected in the sera. These data support continued clinical investigation of the protection against infections in cystic fibrosis patients and patients prone to P. aeruginosa infections.

[Surgery of acquired laryngotracheal stenoses in childhood. Experiences and results from 1988-1998. II: The cricotracheal resection]. / Die Chirurgie der erworbenen laryngotrachealen Stenosen im Kindesalter. Erfahrungen und Ergebnisse von 1988-1998. Teil II: Die cricotracheale Resektion.

Approximately 90% of infants and children with severe acquired laryngotracheal stenoses are tracheotomy dependent and therefore impaired in their physical and speech developments. In addition, tracheotomized infants can be endangered by the cannula due to the possible crusting of secretions or its dislocation. Thus, early repair of a stenosis is mandatory. Within the last 10 years, we successfully operated on 18 children with severe laryngotracheal stenoses. Ten children were treated with a modified Cotton technique. This paper reports our results of cricotracheal resection performed in 8 children since 1994 (age distribution: 7 months through age 15 years). Four children had Cotton grade II stenoses, three had grade III stenoses and one grade IV stenoses. In 3 patients a tracheotomy had been performed at another institution. Since their tracheostomas were too far caudal, they could not be included in the primary resection. All 8 children have been successfully decannulated. Five children without tracheotomies could be extubated uneventfully on the 5th postoperative day. All three primarily tracheotomized children needed further endotracheal stenting with T-tubes because of stomal and suprastomal collapse. Two of these latter children additionally required a tracheoplasty with rib cartilage grafts in order to stabilize the suprastomal trachea prior to decannulation. No patient experienced injuries to the recurrent laryngeal nerves or insufficiencies of the anastomosis. All children's voices were not impaired. This is the third report in literature of cricotracheal resections in infants and children, indicating that this effective, one-stage procedure is superior to laryngotracheal reconstruction with rib cartilage.

[Surgery of acquired laryngotracheal stenoses in childhood. Experiences and results from 1988 to 1998. I: Laryngotracheal reconstruction]. / Die Chirurgie der erworbenen laryngotrachealen Stenosen im Kindesalter. Erfahrungen und Ergebnisse von 1988 bis 1998. Teil I: Die Laryngotracheale Rekonstruktion.

Subglottic laryngotracheal stenosis represents the most severe intubation injury and is increasingly encountered in children due to long-term ventilation during intensive care treatment. Since more than 90% of these children have tracheostomies their physical, psychosocial and speech development can be greatly impaired. A tracheostomy in infants can also be a potentially life-threatening condition, making necessary resolution of the laryngotracheal stenosis and removal of the tracheostoma as soon as possible. During the past 10 years, we have treated 46 children with laryngotracheal problems, including 18 children with severe laryngotracheal stenosis. Ten children (3 with grade II stenosis and 7 with grade III stenosis) were treated by laryngotracheal reconstruction using an anterior rib cartilage graft as described by Cotton. One child with posterior glottic stenosis required a posterior laminotomy with a second rib cartilage graft. Differing from the original method, we stabilized the enlarged endotracheal lumen postoperatively with a Montgomery t-tube. This was kept in place for 10 months on average (shortest period, 6 months; longest period, 12 months). All 10 children could be decannulated, and the tracheostoma closed. Three of the children were operated in other institutions and had a different technique prior to our intervention. Two of our operations failed initially. However, both patients were treated successfully by a second intervention (which was the fourth operation for one of the patients). The reasons for our modification, the operative technique and tips for postoperative management, as well as possible pitfalls and complications, are discussed.

[Experiences with distraction osteogenesis in therapy of severe peripheral airway obstruction in infancy and early childhood]. / Erfahrungen mit der Distraktionsosteogenese bei der Therapie schwerer peripherer Atemwegobstruktionen im Säuglings- und Kleinkindalter.

Children with craniofacial malformations are at special risk for the development of peripheral airway obstruction. The problems are magnified in patients with retroposition or hypoplasia of the mandible. In these cases, the base of the tongue is posteriorly displaced, hereby decreasing the airway diameter. By application of distraction osteogenesis the mandible can be lengthened to move the base of the tongue forward and open the airway. Three female patients aged between 7, 11, and 15 months suffering from peripheral airway obstruction caused by mandibular hypoplasia were treated by gradual distraction. All of them had a gastrostomy or a nasogastral tube in place, respectively, due to severe nutrition problems. In the youngest patient tracheostomy was performed shortly after birth and was already planned in the 15-month-old child, who had received a permanent nasopharyngeal tube. The 11-month-old child suffered from severe refractory sleep apnea. Exercises in oral feeding were possible in the youngest patient after 10 days of distraction. In the oldest one, the airway tube was removed on the six day of distraction and, thus, tracheotomy was successfully avoided. In the 11-month-old child apneic events a rapidly decreased. Our experience suggests that distraction osteogenesis after careful preoperative evaluation can be successfully performed for the treatment of peripheral airway obstruction in patients with selected craniofacial anomalies.

The role of circulating food antigen-specific lymphocytes in food allergic children with atopic dermatitis.

In this study we evaluated antigen-specific in vitro responses of peripheral blood lymphocytes to lipopolysaccharide (LPS)-depleted food allergens in children who reacted to food challenge (cow's milk or hen's egg) with a deterioration of their atopic dermatitis (AD). Some of the children showed immediate symptoms (urticaria, bronchial asthma or gastrointestinal symptoms) as well. The proliferation of casein-stimulated lymphocytes from children reacting to cow's milk (age 0.7-5.9 years) was significantly higher (P < 0.01) than the proliferation of lymphocytes from 15 children with AD without milk allergy (age: 2.1-9.1 years). Twenty-eight T-cell clones (TCC) were established from the blood of three children sensitized to cow's milk and hen's egg who reacted to double-blind, placebo-controlled oral food challenge both with a deterioration of AD and with immediate symptoms. Surprisingly, 16 of 28 casein- or ovalbumin-specific TCC were CD8+. All TCC produced high amounts of IFN-gamma upon stimulation with concanavalin A. In addition, 75% of the CD4+ TCC and 44% of the CD8+ TCC secreted IL-4. Our results indicate that: (i) food-specific proliferation of blood lymphocytes can be detected in patients with clinically relevant food allergy with LPS-depleted allergens in vitro and (ii) circulating food-specific lymphocytes are CD4+ and CD8+ T cells with the capacity of producing both type 1 and type 2 cytokines.

Alteration of pulmonary macrophage function by respiratory syncytial virus infection in vitro.

Alveolar macrophages (AL) are the first line of defense against inhaled pathogens and are exposed to virus during the course of a respiratory syncytial virus (RSV) infection. Interference of virus with alveolar macrophage functions may contribute to the risk of acquiring secondary bacterial infections during or after respiratory tract infections with RSV or other viral agents. We studied whether murine AL get infected with RSV and whether they support viral replication in vitro. In addition, the effects of RSV on microbicidal and on immunoregulatory functions were examined. Only a subpopulation of AL expressed viral F proteins after exposure of these cells to RSV. Infected AL released only small amounts of infectious virus into the supernatant. The extent of virus replication in AL seemed to be dependent in part on the amount of IFN induced by the virus, as has been demonstrated by infection of lung tissue macrophages and AL in vitro. In general, RSV infection of pulmonary macrophages appeared to be abortive. Nevertheless, release of reactive oxygen intermediates, phagocytosis, and killing of protozoa were reduced in RSV-infected AL in comparison to noninfected AL. In contrast, RSV stimulated secretion of TNF-alpha, IL-1, and IL-6 in an infectious-dose dependent manner. Along with the increased cytokine release, accessory functions of AL were increased after RSV exposure. Thus, exposure of AL to RSV appeared to stimulate their immunoregulatory functions, whereas the microbicidal activity of these cells seemed to be severely diminished.

Interaction of alveolar macrophages and respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract illness in infants. However, the mechanisms leading to resolution of RSV infections are poorly understood. Since alveolar macrophages play an important role in defending the respiratory tract against infectious agents we investigated the interactions of RSV with these cells. Murine alveolar macrophages were challenged in vitro with RSV at different multiplicities of infection. The percentage of macrophages expressing viral antigen was determined by staining with monoclonal anti-RSV antibodies and evaluation by fluorescence microscopy or FACS analysis. The ability of macrophages to support virus replication was measured by a plaque forming assay on HEp-2 cells. Cell lysates of macrophages contained only small amounts of viable RSV in comparison to disrupted HEp-2 cells. The amount of viable RSV as well as the percentage of macrophages expressing viral antigen decreased rapidly over time. Activated macrophages had a reduced virus load in comparison to resting macrophages. RSV infected macrophages released biologically active tumor necrosis factor (TNF) in a virus dose dependent manner. In contrast, a high virus inoculum resulted in reduced microbicidal activity and oxygen radical production. Our results suggest that RSV infection influences different functions of alveolar macrophages in various ways. Since TNF is thought to restrict viral replication in several cell types it may play a role in limiting virus replication.