Nod1 signaling overcomes resistance of S. pneumoniae to opsonophagocytic killing

Airway infection by the Gram-positive pathogen Streptococcus pneumoniae (Sp) leads to recruitment of neutrophils but limited bacterial killing by these cells. Co-colonization by Sp and a Gram-negative species, Haemophilus influenzae (Hi), provides sufficient stimulus to induce neutrophil and complement-mediated clearance of Sp from the mucosal surface in a murine model. Products from Hi, but not Sp, also promote killing of Sp by ex vivo neutrophil-enriched peritoneal exudate cells. Here we identify the stimulus from Hi as its peptidoglycan. Enhancement of opsonophagocytic killing was facilitated by signaling through nucleotide-binding oligomerization domain-1 (Nod1), which is involved in recognition of gamma-D-glutamyl-meso-diaminopimelic acid (meso-DAP) contained in cell walls of Hi but not Sp. Neutrophils from mice treated with Hi or compounds containing meso-DAP, including synthetic peptidoglycan fragments, showed increased Sp killing in a Nod1-dependent manner. Moreover, Nod1(-/-) mice showed reduced Hi-induced clearance of Sp during co-colonization. These observations offer insight into mechanisms of microbial competition and demonstrate the importance of Nod1 in neutrophil-mediated clearance of bacteria in vivo.     

Modulation of airway inflammation and bacterial clearance by epithelial cell ICAM-1

Many cell types in the airway express the adhesive glycoprotein for leukocytes intercellular adhesion molecule-1 (ICAM-1) constitutively and/or in response to inflammatory stimuli. In this study, we identified functions of ICAM-1 on airway epithelial cells in defense against infection with Haemophilus influenzae. Initial experiments using a mouse model of airway infection in which the bacterial inoculum was mixed with agar beads that localize inflammation in airways demonstrated that ICAM-1 expression was required for efficient clearance of H. influenzae. Airway epithelial cell ICAM-1 expression required few or no leukocytes, suggesting that epithelial cells could be activated directly by interaction with bacteria. Specific inhibition of ICAM-1 function on epithelial cells by orotracheal injection of blocking antibodies resulted in decreased leukocyte recruitment and H. influenzae clearance in the airway. Inhibition of endothelial cell ICAM-1 resulted in a similar decrease in leukocyte recruitment but did not affect bacterial clearance, indicating that epithelial cell ICAM-1 had an additional contribution to airway defense independent of effects on leukocyte migration. To assess this possibility, we used an in vitro model of neutrophil phagocytosis of bacteria and observed significantly greater engulfment of bacteria by neutrophils adherent to epithelial cells expressing ICAM-1 compared with nonadherent neutrophils. Furthermore, bacterial phagocytosis and killing by neutrophils after interaction with epithelial cells were decreased when a blocking antibody inhibited ICAM-1 function. The results indicate that epithelial cell ICAM-1 participates in neutrophil recruitment into the airway, but its most important role in clearance of H. influenzae may be assistance with neutrophil-dependent bacterial killing.     

Minimization of bacterial size allows for complement evasion and is overcome by the agglutinating effect of antibody

The complement system, which functions by lysing pathogens directly or by promoting their uptake by phagocytes, is critical for controlling many microbial infections. Here, we show that in Streptococcus pneumoniae, increasing bacterial chain length sensitizes this pathogen to complement deposition and subsequent uptake by human neutrophils. Consistent with this, we show that minimizing chain length provides wild-type bacteria with a competitive advantage in vivo in a model of systemic infection. Investigating how the host overcomes this virulence strategy, we find that antibody promotes complement-dependent opsonophagocytic killing of Streptococcus pneumoniae and lysis of Haemophilus influenzae independent of Fc-mediated effector functions. Consistent with the agglutinating effect of antibody, F(ab')(2) but not Fab could promote this effect. Therefore, increasing pathogen size, whether by natural changes in cellular morphology or via antibody-mediated agglutination, promotes complement-dependent killing. These observations have broad implications for how cell size and morphology can affect virulence among pathogenic microbes.     

Stimulation of bacterial adherence by neutrophil defensins varies among bacterial species but not among host cell types

Adherence of Haemophilus influenzae to bronchial epithelial cells is enhanced by neutrophil defensins, which are released from activated neutrophils during inflammation [Gorter et al. (1998) J. Infect. Dis. 178, 1067-1078]. In this study, we showed that the adherence of H. influenzae to various epithelial, fibroblast-like and endothelial cell types was significantly enhanced by defensins (20 microg ml(-1)). Defensins stimulated also the adherence of Moraxella catarrhalis, Neisseria meningitidis and nonencapsulated Streptococcus pneumoniae to the NCI-H292 cell line. In contrast, defensins did not affect the adherence of Pseudomonas aeruginosa, encapsulated S. pneumoniae, Escherichia coli and Staphylococcus epidermidis. These results suggest that the defensin-enhanced adherence might support the adherence and possibly persistence of the selected bacterial species using the respiratory tract as port of entry.     

Nod1 mediates cytoplasmic sensing of combinations of extracellular bacteria

During mucosal colonization, epithelial cells are concurrently exposed to numerous microbial species. Epithelial cytokine production is an early component of innate immunity and contributes to mucosal defence. We have previously demonstrated a synergistic response of respiratory epithelial cells to costimulation by two human pathogens, Streptococcus pneumoniae and Haemophilus influenzae. Here we define a molecular mechanism for the synergistic activation of epithelial signalling during polymicrobial colonization. H. influenzae peptidoglycan synergizes with the pore-forming toxin pneumolysin from S. pneumoniae. Radiolabelled peptidoglycan enters epithelial cells more efficiently in the presence of pneumolysin, consistent with peptidoglycan gaining access to the cytoplasm via toxin pores. Other pore-forming toxins (including anthrolysin O from Bacillus anthracis and Staphylococcus aureus alpha-toxin) can substitute for pneumolysin in the generation of synergistic responses. Consistent with a requirement for pore formation, S. pneumoniae expressing pneumolysin but not an isogenic mutant expressing a non-pore-forming toxoid prime epithelial responses. Nod1, a host cytoplasmic peptidoglycan-recognition molecule, is crucial to the epithelial response. Taken together, these findings demonstrate a role for cytosolic recognition of peptidoglycan in the setting of polymicrobial epithelial stimulation. We conclude that combinations of extracellular organisms can activate innate immune pathways previously considered to be reserved for the detection of intracellular microorganisms.     

Induction of murine B cell proliferation and immunoglobulin synthesis by some bacterial ribosomes

Ribosomal preparations from Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, and Streptococcus pneumoniae were investigated with respect to their activating capacity towards murine lymphoid cells. The proliferation of BALB/c spleen cells was induced in a dose-dependent fashion (from 1 to 100 micrograms/ml) by ribosomes of K. pneumoniae, H. influenzae, and S. pyogenes with a peak activity at 48 or 72 hr of culture. The majority of the blast cells induced by these ribosomal preparations were positive for surface-immunoglobulin (S-Ig) and negative for Thy 1.2. Furthermore, K. pneumoniae, H. influenzae, and S. pyogenes ribosomes induced the synthesis of IgM and some IgA. Cell proliferation and induction of IgM production were also demonstrated with the 3 ribosomal preparations using spleen cells from athymic nude (nu+/nu+) mice, Lyb-5-defective CBA/N spleen cells, B cell-enriched and T cell-depleted BALB/c spleen cell suspensions, as well as spleen cells from the Ips gene-deficient C3H/HeJ strain. Cell culture supernatants contained specific anti-ribosome IgM antibodies. Antibodies of other specificities (anti-sheep erythrocytes) were also demonstrated in supernatants from K. pneumoniae-stimulated cultures. Evidence against a possible role of contamination of K. pneumoniae and H. influenzae ribosomes by lipopolysaccharide- or lipid A-associated proteins in this effect is discussed. Ribosomes from S. pneumoniae did not induce 3H-thymidine incorporation nor Ig production. None of the 4 ribosomal preparations was found to stimulate T cell blastogenesis or to induce interleukin-2 production by naive BALB/c spleen cells. Finally, ribosomes from H. influenzae, S. pyogenes, S. pneumoniae but not those of K. pneumoniae stimulated interleukin-1 production by adherent spleen cells, from BALB/c mice.     

Role of galectin-3 in leukocyte recruitment in a murine model of lung infection by Streptococcus pneumoniae

Pneumonia can be caused by a variety of pathogens, among which Streptococcus pneumoniae causes one of the most common forms of community-acquired pneumonia. Depending on the invading pathogen, the elements of the immune response triggered will vary. For most pathogens, such as Escherichia coli, neutrophil recruitment involves a well-described family of adhesion molecules, beta(2)-integrins. In the case of streptococcal pneumonia, however, neutrophil recruitment occurs mainly through a beta(2)-integrin-independent pathway. Despite decades of research on this issue, the adhesion molecules involved in neutrophil recruitment during lung infection by S. pneumoniae have not been identified. We have previously shown that galectin-3, a soluble mammalian lectin, can be found in lungs infected by S. pneumoniae, but not by E. coli, and can mediate the adhesion of neutrophils on the endothelial cell layer, implying its role in the recruitment of neutrophils to lungs infected with S. pneumoniae. In this study, using galectin-3 null mice, we report further evidence of the involvement of this soluble lectin in the recruitment of neutrophils to S. pneumonia-infected lungs. Indeed, in the absence of galectin-3, lower numbers of leukocytes, mainly neutrophils, were recruited to the infected lungs during infection by S. pneumoniae. In the case of beta(2)-integrin-dependent recruitment induced by lung infection with E. coli, the number of recruited neutrophils was not reduced. Thus, taken together, our data suggest that galectin-3 plays a role as a soluble adhesion molecule in the recruitment of neutrophils to lungs infected by S. pneumoniae, which induces beta(2)-integrin-independent migration.     

Rab27a Is Essential for the Formation of Neutrophil Extracellular Traps (NETs) in Neutrophil-Like Differentiated HL60 Cells

Neutrophils play a crucial role in host defence. In response to a variety of inflammatory stimulation, they form neutrophil extracellular traps (NETs). NETs are extracellular structures composed of chromatin fibers decorated with antimicrobial proteins and developing studies indicate that NETs contribute to extracellular microbial killing. While the intracellular signaling pathways that regulate NET formation remain largely unknown, there is growing evidence that generation of reactive oxygen species (ROS) is a key event for NET formation. The Rab family small GTPase Rab27a is an important component of the secretory machinery of azurophilic granules in neutrophils. However, the precise mechanism of NET formation and whether or not Rab27a contributes to this process are unknown. Using neutrophil-like differentiated HL60 cells, we show here that Rab27a plays an essential role in both phorbol myristate acetate (PMA)- and Candida albicans-induced NET formation by regulating ROS production. Rab27a-knockdown inhibited ROS-positive phagosome formation during complement-mediated phagocytosis. To investigate the role of Rab27a in neutrophil function in detail, both primary human neutrophils and neutrophil-like differentiated HL60 cells were treated with PMA, and NET formation process was assessed by measurement of release of histone H3 into the medium, citrullination of the arginine in position 3 of histone H4 and chase of the nuclear change of the living cells in the co-existence of both cell-permeable and -impermeable nuclear indicators. PMA-induced NET formation occured sequentially in both neutrophil-like differentiated HL60 cells and primary neutrophils, and Rab27a-knockdown clearly inhibited NET formation in association with reduced ROS production. We also found that serum-treated Candida albicans triggers NET formation in a ROS-dependent manner, and that Rab27a-knockdown inhibits this process as well. Our findings demonstrate that Rab27a plays an important role in NET formation induced by both Candida albicans infection and PMA treatment by regulating ROS production.     

Etiology of acute bronchitis in young children

In the microbiological examination of 132 children aged 0-3 years with acute bronchitis, Streptococcus pneumoniae and Haemophilus influenzae at a concentration of greater than or equal to 10(4) cells/ml, as well as different species of opportunistic bacteria, were isolated from tracheobronchial washings obtained from 100 of these children. S. pneumoniae and H. influenzae were found to play the leading role in the etiology of the acute bacterial inflammatory process in acute bronchitis in children.     

IgA1 proteases from Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, and Streptococcus sanguis: comparative immunochemical studies

IgA1 proteases from H. influenzae, N. meningitidis, S. pneumoniae, and S. sanguis were compared with respect to site of cleavage in the IgA1 molecule and EDTA sensitivity. Proteases from S. sanguis and S. pneumoniae cleaved the Pro (227)-Thr (228) bond within the hinge region of the alpha 1 chain and were inhibited by EDTA. H. influenzae IgA1 protease cleaved the Pro (231)-Ser (232) peptide bond. The activity of IgA1 proteases from H. influenzae and N. meningitidis was unaffected by EDTA. Purified and denatured alpha 1 chain was cleaved only in the hinge region. Other component chains of secretory IgA (secretory component, light and J chains) were not susceptible. In addition to IgA1 protease, S. pneumoniae released exo- and endoglycosidases that removed a considerable portion of carbohydrate side chains of IgA1; this activity was absent from crude IgA1 protease preparations of the other three bacterial species. Association in vitro of polymeric IgA1 with SC did not inhibit the degradation of IgA1 proteases. The considerable resistance of secretory IgA to cleavage by IgA1 proteases may be explained in part by the presence of IgA1 protease-neutralizing antibodies in secretory IgA.     

Factors contributing to hydrogen peroxide resistance in Streptococcus pneumoniae include pyruvate oxidase (SpxB) and avoidance of the toxic effects of the fenton reaction

Aerobic growth of Streptococcus pneumoniae results in production of amounts of hydrogen peroxide (H(2)O(2)) that may exceed 1 mM in the surrounding media. H(2)O(2) production by S. pneumoniae has been shown to kill or inhibit the growth of other respiratory tract flora, as well as to have cytotoxic effects on host cells and tissue. The mechanisms allowing S. pneumoniae, a catalase-deficient species, to survive endogenously generated concentrations of H(2)O(2) that are sufficient to kill other bacterial species is unknown. In the present study, pyruvate oxidase (SpxB), the enzyme responsible for endogenous H(2)O(2) production, was required for survival during exposure to high levels (20 mM) of exogenously added H(2)O(2). Pretreatment with H(2)O(2) did not increase H(2)O(2) resistance in the mutant, suggesting that SpxB activity itself is required, rather than an H(2)O(2)-inducible pathway. SpxB mutants synthesized 85% less acetyl-phosphate, a potential source of ATP. During H(2)O(2) exposure, ATP levels decreased more rapidly in spxB mutants than in wild-type cells, suggesting that the increased killing of spxB mutants was due to more rapid ATP depletion. Together, these data support the hypothesis that S. pneumoniae SpxB contributes to an H(2)O(2)-resistant energy source that maintains viability during oxidative stress. Thus, SpxB is required for resistance to the toxic by-product of its own activity. Although H(2)O(2)-dependent hydroxyl radical production and the intracellular concentration of free iron were similar to that of Escherichia coli, killing by H(2)O(2) was unaffected by iron chelators, suggesting that S. pneumoniae has a novel mechanism to avoid the toxic effects of the Fenton reaction.     

Role of p38 MAP kinase and transforming growth factor-beta signaling in transepithelial migration of invasive bacterial pathogens

Streptococcus pneumoniae and Haemophilus influenzae are human pathogens that often asymptomatically colonize the mucosal surface of the upper respiratory tract, but also occasionally cause invasive disease. The ability of these species to traverse the epithelium of the airway mucosa was modeled in vitro using polarized respiratory epithelial cells in culture. Migration across the epithelial barrier was preceded by loss of transepithelial resistance. Membrane products of S. pneumoniae that included lipoteichoic acid induced disruption of the epithelial barrier in a Toll-like receptor 2-dependent manner. This result correlates with a recent genetic study that associates increased TLR2 signaling with increased rates of invasive pneumococcal disease in humans. Loss of transepithelial resistance by the TLR2 ligand correlated with activation of p38 MAP kinase and transforming growth factor (TGF)-beta signaling. Activation of p38 MAPK and TGF-beta signaling in epithelial cells upon nasal infection with S. pneumoniae was also demonstrated in vivo. Inhibition of either p38 MAPK or TGF-beta signaling was sufficient to inhibit the migration of S. pneumoniae or H. influenzae. Our data shows that diverse bacteria utilize common mechanisms, including MAPK and TGF-beta signaling pathways to disrupt epithelial barriers and promote invasion.     

Different meningitis-causing bacteria induce distinct inflammatory responses on interaction with cells of the human meninges

The interactions of bacterial pathogens with cells of the human leptomeninges are critical events in the progression of meningitis. An in vitro model based on the culture of human meningioma cells was used to investigate the interactions of the meningeal pathogens Escherichia coli K1, Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae. A rank order of association with meningioma cells was observed, with N. meningitidis showing the highest levels of adherence, followed by E. coli, S. pneumoniae and H. influenzae. Neisseria meningitidis and H. influenzae did not invade meningioma cells or induce cell death, but induced a concentration-dependent secretion of inflammatory mediators. Neisseria meningitidis induced higher levels of IL-6, MCP-1, RANTES and GM-CSF than H. influenzae, but there was no significant difference in the levels of IL-8 induced by both pathogens. Streptococcus pneumoniae was also unable to invade meningioma cells, but low concentrations of bacteria failed to stimulate cytokine secretion. However, higher concentrations of pneumococci led to cell death. By contrast, only E. coli K1 invaded meningioma cells directly and induced rapid cell death before an inflammatory response could be induced. These data demonstrate that the interactions of different bacterial pathogens with human meningeal cells are distinct, and suggest that different intervention strategies may be needed in order to prevent the morbidity and mortality associated with bacterial meningitis.     

NADPH Oxidase-Dependent Production of Reactive Oxygen Species Induces Endoplasmatic Reticulum Stress in Neutrophil-Like HL60 Cells

Reactive oxygen species (ROS) primarily produced via NADPH oxidase play an important role for killing microorganisms in neutrophils. In this study we examined if ROS production in Human promyelocytic leukemia cells (HL60) differentiated into neutrophil-like cells (dHL60) induces ER stress and activates the unfolded protein response (UPR). To cause ROS production cells were treated with PMA or by chronic hyperglycemia. Chronic hyperglycemia failed to induce ROS production and did not cause activation of the UPR in dHL60 cells. PMA, a pharmacologic NADPH oxidase activator, induced ER stress in dHL60 cells as monitored by IRE-1 and PERK pathway activation, and this was independent of calcium signaling. The NADPH oxidase inhibitor, DPI, abolished both ROS production and UPR activation. These results show that ROS produced by NADPH oxidase induces ER stress and suggests a close association between the redox state of the cell and the activation of the UPR in neutrophil-like HL60 cells.     

Upper respiratory tract microbial communities, acute otitis media pathogens, and antibiotic use in healthy and sick children

The composition of the upper respiratory tract microbial community may influence the risk for colonization by the acute otitis media (AOM) pathogens Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. We used culture-independent methods to describe upper respiratory tract microbial communities in healthy children and children with upper respiratory tract infection with and without concurrent AOM. Nasal swabs and data were collected in a cross-sectional study of 240 children between 6 months and 3 years of age. Swabs were cultured for S. pneumoniae, and real-time PCR was used to identify S. pneumoniae, H. influenzae, and M. catarrhalis. The V1-V2 16S rRNA gene regions were sequenced using 454 pyrosequencing. Microbial communities were described using a taxon-based approach. Colonization by S. pneumoniae, H. influenzae, and M. catarrhalis was associated with lower levels of diversity in upper respiratory tract flora. We identified commensal taxa that were negatively associated with colonization by each AOM bacterial pathogen and with AOM. The balance of these relationships differed according to the colonizing AOM pathogen and history of antibiotic use. Children with antibiotic use in the past 6 months and a greater abundance of taxa, including Lactococcus and Propionibacterium, were less likely to have AOM than healthy children (odds ratio [OR], 0.46; 95% confidence interval [CI], 0.25 to 0.85). Children with no antibiotic use in the past 6 months, a low abundance of Streptococcus and Haemophilus, and a high abundance of Corynebacterium and Dolosigranulum were less likely to have AOM (OR, 0.51; 95% CI, 0.31 to 0.83). An increased understanding of polymicrobial interactions will facilitate the development of effective AOM prevention strategies.     

Toll-like receptor 4 mediates innate immune responses to Haemophilus influenzae infection in mouse lung.

Toll-like receptors (TLRs) have been implicated in the regulation of host responses to microbial Ags. This study characterizes the role of TLR4 in the innate immune response to intrapulmonary administration of Haemophilus influenzae in the mouse. Two different strains of mice efficiently cleared aerosolized H. influenzae concurrent with a brisk elaboration of IL-1beta, IL-6, TNF-alpha, macrophage-inflammatory protein (MIP)-1alpha, and MIP-2 in bronchoalveolar lavage and a corresponding mobilization of intrapulmonary neutrophils. Congenic strains of mice deficient in TLR4 demonstrated a substantial delay in clearance of H. influenzae with diminished IL-1beta, IL-6, TNF-alpha, MIP-1alpha, and MIP-2 in bronchoalveolar lavage and a notable absence of intrapulmonary neutrophils. In TLR4-expressing animals, but not TLR4-deficient animals, TNF-alpha and MIP-1alpha expression was up-regulated in epithelial cells of the conducting airway in response to H. influenzae which was preceded by an apparent activation of the NF-kappaB pathway in these cells based on the findings of decreased overall IkappaB and an increase in its phosphorylated form. This study demonstrates a critical role of TLR4 in mediating an effective innate immune response to H. influenzae in the lung. This suggests that the airway epithelia might contribute to sensing of H. influenzae infection and signaling the innate immune response.     

Recognition of Streptococcus pneumoniae by the innate immune system

Streptococcus pneumoniae is both a frequent colonizer of the upper respiratory tract and a leading cause of life-threatening infections such as pneumonia, meningitis and sepsis. The innate immune system is critical for the control of colonization and for defence during invasive disease. Initially, pneumococci are recognized by different sensors of the innate immune system called pattern recognition receptors (PRRs), which control most subsequent host defence pathways. These PRRs include the transmembrane Toll-like receptors (TLRs) as well as the cytosolic NOD-like receptors (NLRs) and DNA sensors. Recognition of S. pneumoniae by members of these PRR families regulates the production of inflammatory mediators that orchestrate the following immune response of infected as well as neighbouring non-infected cells, stimulates the recruitment of immune cells such as neutrophils and macrophages, and shapes the adaptive immunity. This review summarizes the current knowledge of the function of different PRRs in S. pneumoniae infection.     

Genetic and functional analysis of the phosphorylcholine moiety of commensal Neisseria lipopolysaccharide

Phosphorylcholine (ChoP) is a common surface feature of many mucosal organisms, including Neisseria spp., in which it is present exclusively on pili of pathogenic Neisseria and on the lipopolysaccharide (LPS) of commensal Neisseria (Cn). Its presence in Cn has been confirmed by nuclear magnetic resonance. It appears that choline is the main source for the production of ChoP by Cn. We have sequenced a locus, containing four genes (licA-D) with 47-73% identity to the lic1 locus of Haemophilus influenzae (Hi) and 21-40% identity to lic genes in Streptococcus pneumoniae, involved in the production and incorporation of ChoP. The arrangement of the Cn genes and the presence of CAAT repeats, responsible for phase variation of ChoP expression, resemble Hi and differ from S. pneumoniae. Cn DNA flanking the lic locus contains genes ilvE and NMA2149 with >85% identity to the pathogenic Neisseria genes. However, there are no lic genes in the corresponding location or elsewhere in pathogenic Neisseria. This suggests either the loss of the locus from pathogenic Neisseria or a horizontal transfer of genes to Cn, perhaps from H. influenzae spp. As in Hi, ChoP enhances adherence to and invasion of human epithelial cells via the receptor for platelet-activating factor. However, ChoP expression also increases susceptibility to serum killing mediated by complement and C-reactive protein. Taken together, these observations support the hypothesis that the ability of many organisms to switch off ChoP expression rapidly represents an important adaptation to different environments encountered during the colonization/infection process and that the ChoP moiety apparently synthesized by distinct means in pathogenic and commensal Neisseria represents an advantage in the colonization properties of these bacteria.     

Bacterial infectious process in patients with chronic obliterating bronchiolitis

Chronic obliterating bronchiolitis in children is characterized by the presence of chronic multifactor inflammatory infectious process with Streptococcus pneumoniae and Haemophilus influenzae being the leading causative agents. The inflammatory process is induced and maintained by S. pneumoniae and H. influenzae or by the association of these two pathogens, but the severity of the clinical course of obliterating bronchiolitis is determined by H. influenzae, which is confirmed by its high detection rate at the phase of exacerbation in patients with pronounced bronchial obstruction.