[Measurements of L-lactate and H2O2 in exhaled breath condensate at rest and mild to moderate exercise in young and healthy subjects]. / L-Laktat und H2O2-Bestimmungen im Atemkondensat unter Ruhebedingungen sowie unter leichter bis mittelgradiger Belastung von jungen gesunden Probanden.

BACKGROUND: The recently developed microenzyme detectors make a non-invasive measurement of inflammatory markers and L-lactate in exhaled breath condensate (EBC) possible. In a group of young and healthy subjects, we examined whether L-lactate and H (2)O (2) can be detected in EBC. METHODS: During resting conditions as well as at 60 and 120 Watt external load on a cycle ergospirometer 100 l exhaled air were collected under stationary load conditions from 19 healthy subjects. Exhaled breath condensate (EBC) was obtained by cooling the expired air volume. The analysis was performed within 90 min using an ECo-Check amperometer (Viasys Health Care). The H (2)O (2) measurement was performed amperometrically by means of a biosensor after chemical reaction catalysed by peroxidase. Lactate measurements were performed using a bienzyme sensor after lactate oxidase-induced oxidation of L-lactate to pyruvate and H (2)O (2). The rates of release of L-lactate in nmol/min und H (2)O (2) in pmol/min were calculated from the concentrations of L-lactate and H (2)O (2) in the EBC and the time of collection. RESULTS: At rest 100 l exhaled air were collected in 10.6 +/- 5.1 min, and 0.99 +/- 0.3 ml EBC were obtained, at the 60 Watt step 1.23 +/- 0.47 ml EBC were collected in 6.7 +/- 1.8 min, and at 120 Watt 1.09 +/- 0.38 ml EBC in 4.8 +/- 0.8 min. At rest, there was a mean rate of L-lactate release of 3.3 +/- 3.1 nmol/min, which increased at the 60 Watt step to 8.4 +/- 5.1 nmol/min (p < 0.05), and at 120 Watt to 5.0 +/- 12.6 nmol/min (p < 0.02). The rate of L-lactate was proportional to the metabolic rate (r = 0.99). The rate of H (2)O (2) release at rest was 49.1 +/- 37.9 pmol/min, it increased at 60 Watt to 159 +/- 113 pmol/min (p < 0.05) and decreased at 120 Watt to 96.5 +/- 49.5 pmol/min (p < 0.05). CONCLUSIONS: Significant measurable concentrations of L-lactate and H (2)O (2) in the exhaled breath condensate were found already under resting conditions. During external load, an increase in the L-lactate concentration was found, correlating with the metabolic rate. H (2)O (2) is an inflammatory marker, its concentration in the EBC was markedly increased during the first step of applied external load, but less during the second. A probable correlation between L-lactate concentration in EBC and arterialized blood will be studied in future investigations.

Respiratory syncytial virus-induced chronic bronchiolitis in experimentally infected calves.

Human (RSV) and bovine (BRSV) respiratory syncytial virus cause similar infections of the lower respiratory tract. Therefore, experimentally infected calves are suited to the study of RSV-induced chronic bronchiolitis. Colostrum-fed calves aged 17-24 days were successfully infected with BRSV. BRSV strain 375 was applied as an aerosol on 4 consecutive days. Clinical symptoms were already evident on the 1st day after infection. The calves were necropsied 12 weeks after the first infection. Focal severe chronic bronchiolitis with atelectasis and focal bronchiolitis obliterans were demonstrated. The bronchiolar lumina were filled with secretion. Transmission electron microscopy revealed an alteration of the ciliogenesis and partial loss of cilia. Immunhistochemically virus P protein could still be detected, mainly in the epithelial cells of the inflamed bronchioli.

Increased number of T cells committed to IL-5 production after respiratory syncytial virus (RSV) infection of human mononuclear cells in vitro.

We examined changes in the cytokine profile of T cells induced by in vitro infection with RSV. Isolated mononuclear cells from 27 healthy adults and six infants were infected with RSV at a concentration of 3 MOI (multiplicity of infection). After 48 h cells were restimulated with phorbol ester and ionomycin in the presence of monensin for 5 h. The intracellular expression of viral antigen, the cytokines IL-2, IL-4, IL-5, interferon-gamma (IFN-gamma), and the expression of surface markers were assessed by immunofluorescent staining and flow cytometry. There was a significant (P<0.001) rise of IL-5 expression in RSV-infected cultures in comparison with uninfected cultures from the same individuals, whereas there were no changes in the expression of the other lymphokines. The increase in IL-5 generation depended on viable infectious RSV rather than inactivated virus. RSV infection as well as IL-5 production in T cells were confined to the CD8 subpopulation. However, there was no simultaneous expression of RSV antigen and IL-5. Purified T cells did not show any increase in IL-5 generation. However, when the rate of RSV infection was enhanced in monocytes by means of a specific monoclonal antibody, co-cultured T cells displayed an increase of IL-5 production compared with samples with ordinary low rate RSV infection. It is therefore likely that the increased commitment of lymphocytes to produce IL-5 after RSV infection in vitro is mediated by monocytes or other antigen-presenting cells.

Persistence of airway hyperresponsiveness and viral antigen following respiratory syncytial virus bronchiolitis in young guinea-pigs.

Respiratory syncytial virus (RSV) bronchiolitis in infancy is known to be followed by chronic respiratory symptoms and airway hyperresponsiveness in a subgroup of patients. To further investigate the pathogenesis of RSV-induced chronic airway pathology, we infected young guinea-pigs at 4 weeks of age with RSV applied as an aerosol (n=30), and control guinea-pigs with virus-free culture medium (n=24). Infection was confirmed by positive antibody titre to RSV after 6 weeks, and by typical pathological changes of bronchiolitis after 1 week in six animals from each group. Airway hyperresponsiveness was measured weekly for 5 weeks by histamine challenge, using body-plethysmographic measurement of compressed air (CA). The provocative concentration of histamine producing significant airway obstruction (i.e. CA = 0.1 mL) (PC0.1 mL CA in mg x mL(-1)) was calculated from dose-response curves. Six weeks postinfection, the lungs were investigated for the presence of inflammation and of viral antigen by immunofluorescence and immunohistochemistry using a rabbit hyperimmune serum and monoclonal antibodies. Airway responsiveness was increased in the RSV group 1 week postinfection compared to the control group (PC0.1 mL CA median 2.50 vs >10 mg x mL(-1); p<0.001) and this persisted up to 5 weeks postinfection (PC0.1 mL CA median 1.61 vs >10 mg x mL(-1); p<0.001). During the same period, viral antigen persisted in the lungs of infected animals, although there was less inflammation at 6 weeks postinfection than at 1 week postinfection. In guinea-pigs, respiratory syncytial virus infection of the airways causes persistent airway hyperresponsiveness over a period of at least 5 weeks. During this time, viral antigen, but not inflammation, remains detectable in the lungs and might be responsible for ongoing airway hyperresponsiveness.

Bovine rotavirus strain 678 possesses VP4 of serotype P7[5] specificity.

Bovine rotavirus strain 678 is the first G8 strain of bovine origin but the literature is confusing as to its P type. In this study, two-way cross neutralization between 678 and 0510, a prototype G6P7[5] virus, was shown by plaque-reduction neutralization assays, establishing the P type of 678 as being P7[5]. The P7[5] specificity of 678 VP4 was reinforced by the finding that the VP8* portion of 678 VP4 had the highest amino acid identity with those of P7[5] bovine rotaviruses. Apparent contradiction with previous serological studies relates to intricacy of antigenicity and immunogenicity of UK VP4 in reassortants.

Heparin-dependent attachment of respiratory syncytial virus (RSV) to host cells.

In this study we could demonstrate that heparin (ED50 = 0.32 +/- 0.12 microgram/ml), but not heparan sulphate or chondroitin sulphate C is able to inhibit in vitro infection of cells by respiratory syncytial virus (RSV). In addition, this protective effect of heparin could only be observed, when heparin was present at the time of inoculation. Enzymatic digestion of cell surface glycosaminoglycans with heparinase and heparitinase, but not chondroitin sulphate ABC lyase reduced the effectiveness of RSV-infection. Affinity chromatography experiments, using immobilised heparin further demonstrated that RSV attachment protein G was able to bind specifically to heparin. Therefore heparin-like proteoglycans showed properties required for attachment of RSV to host cells.

Role of sensory neuropeptides in PIV-3-infection-induced airway hyperresponsiveness in guinea pigs.

Viral respiratory tract infections are known to induce transient airway hyper-responsiveness. The role of the nonadrenergic noncholinergic neuropeptide system on virus-induced airway hyperresponsiveness was studied in the guinea pig. Ten guinea pigs were inoculated with parainfluenza 3 virus (PIV-3.2 x 10(6) PFU) by nasal route. 16 animals served as untreated controls. Viral infection was proven by histological changes and by demonstration of viral antigen using immunohistochemical techniques. Four days after inoculation, airway responsiveness to inhaled acetylcholine (ACH) aerosol was measured in anesthetized and tracheotomized guinea pigs. The ACH concentration which produced an increase of 100% in pulmonary resistance (PC100 RI) and in dynamic elastance (PC100 Edyn) was calculated from a 5-step ACH dose-response curve (0.125, 0.25, 0.5, 1.0 and 2.0% ACH). Two further groups of 8 PIV-3-infected guinea pigs and 8 noninfected control animals were pretreated with capsaicin in increasing doses (50, 100, 125 and 150 mg/kg) on 4 consecutive days starting 6 days before virus inoculation. Measurements of airway responsiveness to ACH were performed 4 days after virus inoculation. Another 5 uninfected control animals were pretreated only with the solvent for capsaicin and inoculated with virus-free cell supermatant. PIV-3 infection increased airway responsiveness to ACH compared to noninfected controls [PC100 RI 0.81 vs. > 2.0% ACH (median). p < 0.002 PC100 Edyn 0.52 vs. 1.07% ACH (median), p < 0.01]. In capsaicin-pretreated PIV-3-infected animals, airway hyperresponsiveness was completely prevented compared to the virus-infected group without capsaicin pretreatment (PC100 RI > 2.0 vs. 0.81% ACH, p < 0.01; PC100 Edyn 1.42 vs. 0.52% ACH p < 0.01). As neuropeptide depletion with capsaicin completely prevented the increase in airway constrictory response to ACH following virus infection, we conclude that neuropeptides are effectively involved in PIV-3-induced airway hyperresponsiveness in the guinea pig.

Prevalence of antibodies against HEp-2 cell antigen in infants and children hospitalized with respiratory syncytial virus infection.

Infants with respiratory syncytial virus (RSV) infection were shown to have antibodies against HEp-2 cell antigen present in RSV-antigen preparation used for immunoblot analysis. The prevalence of anti HEp-2 cell antibodies was examined in infants hospitalized for RSV infection (n = 49, median age 121 days) compared to rotavirus infected children (n = 30, median age 114 days) and to healthy controls (n = 20, median age 150 days). The immunoblot analysis with RSV-infected and non-infected HEp-2 cells as antigen revealed the expected age-dependent low prevalence of G protein antibodies and clear seroconversion of N and P protein antibodies. HEp-2 antibody prevalence was higher in RSV antigen-positive infants (33/49) than in rotavirus antigen-positive (5/30) and RSV antigen-negative infants (4/20), respectively (p < 0.001). Anti HEp-2 antibodies were mostly directed against 47, 46, 33, 30 kD antigens. A multiple regression analysis found the following correlations (odds ratio; 95% confidence interval): 42 kD RSV antibodies (N protein) with pneumonia (7.58; 1.43-40), 94 kD RSV antibodies (G protein) with bronchiolitis (0.064; 0.006-0.686). This study shows repeated well-known features of humoral immunity in RSV infection. The data on anti HEp-2 antibodies point to a role for these pre-existing autoreactive antibodies in the pathogenesis of RSV infection.

Morphological studies of the respiratory syncytial virus induced bronchiolitis in experimentally infected calves.

RSV-infections of the lower respiratory tract in infancy and early childhood are the most frequent causes of a hyperreactive bronchial system and obstructive lung disease. Studies concerning the morphological alterations of the bronchial mucosa during an RSV-infection are dependent on an experimental animal model. In this study the alterations of the lower respiratory tract from five infected colostrum-fed calves during the initial stage of the infection are described. BRSV strain 375 was applied as an aerosol on four consecutive days. The animals showed clinical symptoms already on the first day after infection. 7 days after the first infection the calves were necropsied. Lobular distributed atelectasis of the lung were found. The corresponding bronchioli were collapsed. The bronchiolar lumina were filled with a putrid exudate. In the bronchiolar wall a band-like lymphocytic infiltrate was found. By confocal laserscanning microscopy and by scanning electron microscopy intracellular viral components marked by an antibody against the viral P-protein were depicted. The intracellular virus inclusions were arranged along the bundles of filaments of the cytoskeleton. By transmission electron microscopy an alteration of the ciliogenesis and in cases of severe cell damage, cell death could be observed. The morphological findings suggest that the cytoskeleton plays an important role in the development of bronchiolitis.

IL-8 release from human neutrophils by the respiratory syncytial virus is independent of viral replication.

Elevated interleukin-8 levels and a massive accumulation of neutrophils (PMN) are the hallmark of a variety of severe lung diseases. The respiratory syncytial virus (RSV), an important respiratory pathogen, induces interleukin-8 (IL-8) release from human PMN, however, the mechanism is as yet unknown. We analyzed the role of virus uptake, intracellular virus replication, virus attachment, and of virus capsid proteins for the induction of IL-8 (protein + mRNA) in human PMN. Cell supernatants were analyzed for IL-8 release via enzyme-linked immunosorbent assay; cell pellets were analyzed for IL-8-specific mRNA expression and for RSV-specific genomic and RSV-specific mRNA by reverse transcriptase-polymerase chain reaction. Stimulation of human PMN with viable, heat-inactivated, or UV-inactivated RSV [at a multiplicity of infection (m.o.i.) from 0.01 up to 10] induced IL-8 production (protein + mRNA) to a similar degree. Maximal IL-8 release was observed at a m.o.i. of 5-10 after 18-24 h. RSV-specific genomic RNA was present inside PMN up to 24 h independent of whether viable or inactivated RSV was used. Withdrawal of extracellular viable or inactivated (heat, UV) RSV after infection of PMN (2 h) abolished IL-8 mRNA expression and IL-8 release; the intracellular persistence of RSV lasted for up to 24 h. Stimulation of human PMN with purified RSV G-protein, a major capsid protein, in a concentration range from 0.1 up to 2.5 microg/5 X 10(5) PMN resulted in an increased IL-8 release from human PMN but to a significantly lesser degree compared with the intact RSV. RSV G-protein concentration above 1 microg inhibited the RSV-induced IL-8 release by up to 90%. Our data contribute to the understanding of the pathomechanisms leading to IL-8 release from human PMN.

Respiratory syncytial virus infection: its role in aeroallergen sensitization during the first two years of life.

Our aim was to study the influence of infection with the respiratory syncytial virus (RSV) in non-hospitalized infants on sensitization to aeroallergens and the early manifestation of atopy. Six hundred and nine infants from the prospective German Multicenter Cohort Study on Atopy were included, 38% of whom had an elevated atopic risk. RSV IgG and IgM antibodies were tested by ELISA with gradient purified RSV antigen. Specific IgE against mites, cat dandruff, birch and grass pollens and relevant nutritional antigens were tested with CAP-RAST-FEIA (Pharmacia, Sweden). Of the cord sera 99% were positive for RSV-IgG, 44.7% at one year and 64.2% (n = 265) at two years of age. The positivity rate after 12 months varied with the season of birth, the number of siblings and the degree of exposure to tobacco smoke; and correlated closely with attacks of wheezing during infancy. Twenty (2.8%) children were found to be sensitized against at least one aeroallergen at one year, and 28 (10.5%) at two years. By the first birthday, mite sensitization (n = 3) could only be seen in the RSV-infected children; grass pollen sensitization (n = 9) was associated with RSV seropositivity (logistic regression model including the confounders mentioned above: with RSV IgG < p = 0.048 > and IgM < p = 0.0006 >), as was birch sensitization (n = 5) with RSV IgM (p = 0.009). No such differences could be detected at two years. No correlation of RSV seropositivity to any allergic manifestation could be found. We conclude, that it is only in the first year of life, that RSV infection plays a significant role in promoting sensitization against aeroallergens, which do not at this time produce allergic symptoms.

Respiratory syncytial virus G-protein modulates cytokine release from human peripheral blood mononuclear cells.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in children and adults. In vivo the host has to cope with intact replicative virus, with non-replicative virus, and/or with viral structural proteins including the outer membrane G-protein. We analyzed the role of purified RSV G-protein with regard to its modulatory efficacy for interleukin (IL) -10, IL-12, and tumor necrosis factor-alpha (TNF-alpha) release from human peripheral blood mononuclear cells (PBMC). These cytokines seem to contribute to the deleterious effect in viral infections. Treatment of PBMC with RSV at a multiplicity of infection of 10 down to 0.001 induced the release of TNF-alpha, IL-10, and IL-12; also time kinetics and dose-responses differed markedly. Stimulation of PBMC with purified RSV G-protein (from 0.001 up to 10 microgram/10(6) PBMC) led only to a pronounced increase in IL-10 within a concentration range from 0.01 up to 0.5 microgram/10(6) PBMC with a maximum between 12 and 18 h of incubation. AT later time points (24, 48, and 72 h) G-protein concentrations above 1 microgram/10(6) PBMC suppressed IL-10, TNF-alpha, and IL-12 release from human PBMC. Coating of PBMC with RSV G-protein suppressed IL-10, TNF-alpha, and IL-12 release after subsequent stimulation with RSV. Our data indicate a regulatory role of RSV G-protein immune responses toward viral infection.

A model for respiratory syncytial virus (RSV) infection based on experimental aerosol exposure with bovine RSV in calves.

Five conventionally kept calves aged between 17 and 24 days were experimentally infected with bovine respiratory syncytial virus (BRSV) by aerosol in order to mimic the natural infection route. The calves were killed and autopsies performed 7 days after the first virus challenge. The BRSV isolate used induced tracheitis, bronchitis and atelectasis in infected calves. The only virus which could be isolated from the lungs of the calves was BRSV. In addition, Mycoplasma bovirhinis was isolated from the lungs or/and trachea of two calves. The clinical and histopathological findings, as well as the detection of BRSV antigens by immunofluorescence in the epithelial cells of lung and trachea, and the reisolation of the virus from bronchoalveolar lavage fluids of all inoculated calves, provided confirmation of successful infection with BRSV.

Detection of respiratory syncytial virus (RSV) antigen in the lungs of guinea pigs 6 weeks after experimental infection and despite of the production of neutralizing antibodies.

Infections with respiratory syncytial virus (RSV) are characterized by frequently occurring reinfections and are regarded to be responsible for bronchial hyperreactivity. In this report we describe a small-animal model suited to study RSV-induced pathogenesis and immune response. Guinea pigs are infected by inhalation of an RSV-aerosol. Lungs of infected animals show signs of a bronchiolitis at 7 days after the initial infection. Although neutralizing serum antibodies are synthesized viral proteins are still detectable at 6 weeks post infection. Therefore, the presence of neutralizing antibodies is obviously not sufficient for rapid clearance of persistent RSV-proteins from the lungs of infected guinea pigs.

The humoral immune response of children and infants to an RSV infection: its maturation and association with illness.

In this study the immune response of 29 newborns and infants hospitalized on behalf of a RSV infection was evaluated. Acute phase and convalescent sera were examined by a neutralization assay, in an immunoblot, and in an ELISA based on 10 synthetic peptides derived from RSV proteins. The last two tests allow to monitor the synthesis of RSV specific antibodies of the infant. Despite the presence of maternal antibodies the initial immune response seems to be random and favours linear epitopes of the protein backbone of viral proteins. The earliest protecting antibodies directed against glycosylated epitopes are acquired in the second half year of life. Antibodies generated during the primary immune response seem to predispose infected children to get ill.

[Models of the cause of spongiform encephalopathies]. / Modelle zur Ursache spongiformer Enzephalopathien.

Transmissible spongiform encephalopathies seem to contradict a dogma in microbiology. There is now increasing evidence that the infectious agents are proteins (prion proteins). These proteins seem to be able to catalyze conformational conversions of a host-encoded isoform. The altered conformation induces intracellular accumulation and may lead to polymerization into fibrils and amyloid rods. Catalytical interactions of infectious prion proteins and their cellular isoforms are dependent on the primary structure. These considerations may be helpful to evaluate the risk of transmission of BSE to humans.

Nonstructural protein 2 (NS2) of respiratory syncytial virus (RSV) detected by an antipeptide serum.

The human respiratory syncytial virus (RSV) is often associated with airway obstruction and is suspected to induce bronchial hyperreactivity. Interactions of viral proteins with cellular components may be responsible for epithelial damage leading to bronchial hyperreactivity. In this study, we describe the localization of the 14.7-kD nonstructural protein 2 (NS2) in RSV-infected cells. The detection of NS2 was performed using antipeptide antibodies elicited against amino acids 109-123 of the predicted sequence of the NS2 protein. By using recombinant NS2, we could clearly demonstrate the specificity of the antipeptide antibodies. With this defined tool, NS2 could be first detected in infected HEp-2 cells at 10 h p.i. subsequently to the detection of N protein. In double-staining experiments, colocalization of NS2, P protein and N protein was demonstrated. The antipeptide antibodies recognized the NS2 protein in the sediment of RSV-infected HEp-2 cells lysed with RIPA buffer at 48 h p.i. The results agree with the reported interaction of RSV with cytoskeletal intermediate filaments. These interactions may implicate essential cellular functions suspected to induce bronchial hyperreactivity.

Studies on the single-shelled rotavirus receptor with a synthetic peptide derived from the cytoplasmic domain of NS28.

The nonstructural glycoprotein NS28 of rotaviruses plays an important part in the assembly of double-shelled rotaviruses. C-terminal domains of the protein function as a receptor for single-shelled rotavirus particles at the membrane of the rough endoplasmic reticulum. In the present report we describe studies performed with a synthetic peptide corresponding to amino acid (aa) 160 to 169, the most hydrophilic C-terminal epitope of NS28. An antipeptide serum raised against this peptide demonstrated that this epitope was accessible in infected MA104 cells. Moreover, polymeric peptide was demonstrated to aggregate single-shelled rotavirus particles. This aggregation could be almost completely inhibited by preincubation with monomeric peptide. Our results clearly demonstrate that the epitope corresponding to aa 160-169 is able to bind single-shelled rotavirus particles.

[Effect of the humoral immune response on the pathogenesis of respiratory syncytial virus (RSV) infection]. / Beitrag der humoralen Immunantwort zur Pathogenese der Respiratory-Syncytial-Virus-(RSV-)Infektion.

Direct cytopathic effects and components of the humoral immune response may contribute to the pathogenesis of respiratory syncytial virus (RSV) infections. Using synthetic peptides as an analytical tool, it could be demonstrated that the primary immune response of young children is directed against other epitopes compared to the situation in adults. RSV-specific antibodies are not only considered to possess protective properties but also to contribute to the pathogenesis of the infection. In our experiments, human adherent cells from lavage could be infected by RSV more effectively in the presence of murine monoclonal antibodies specific for RSV surface proteins.

[Cat sera neutralize rotaviruses of serotype G3]. / Katzenseren neutralisieren Rotaviren des Serotyps G 3.

Group A rotaviruses play an important role for the induction of gastroenteritis. Seroepidemiological studies evaluating the situation in humans have been performed previously. In this study data concerning the importance of group A rotavirus infections and the contribution of the most important serotypes are given for the domestic cat. 91% of the observed sera showed antibodies neutralizing serotype G 3. Antibodies with neutralizing properties directed against other important human serotypes could not be detected. The results obtained are discussed with respect to the formation of reassortant rotaviruses.