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Article Reference Oral bacterial vaccines for the prevention of acute exacerbations in chronic obstructive pulmonary disease and chronic bronchitis.
Oral vaccines using killed bacterial extracts have been used to prevent acute exacerbations of chronic obstructive pulmonary disease (COPD); however, they are not recommended by current clinical guidelines. Two systematic reviews have been published on the efficacy of oral vaccines. The first, on the effects of an oral whole-cell nontypeable Haemophilus influenzae vaccine (NTHi) found a significant decrease in the incidence of acute episodes of chronic bronchitis (Poisson rate ratio 0.666; 95% confidence interval (CI) 0.500, 0.887; P = 0.005), and a 58% reduction in the prescription of antibiotics 3 months after vaccination. The second review evaluated studies that used multicomponent vaccines. It found that the duration of exacerbations was significantly shorter in the treatment group (weighted mean difference -2.7 days, 95% CI -3.5 to -1.8). These reviews suggest that oral vaccines reduce the number, severity, duration, or both, of acute exacerbations. However, many of the primary trials on which they are based are small and methodologically flawed. Further trials are needed before the use of oral vaccines could be considered as part of the routine clinical management of patients with COPD or chronic bronchitis.
Article Reference Identification of biofilm proteins in non-typeable Haemophilus Influenzae.
Non-typeable Haemophilus influenzae biofilm formation is implicated in a number of chronic infections including otitis media, sinusitis and bronchitis. Biofilm structure includes cells and secreted extracellular matrix that is ``slimy'' and believed to contribute to the antibiotic resistant properties of biofilm bacteria. Components of biofilm extracellular matrix are largely unknown. In order to identify such biofilm proteins an ex-vivo biofilm of a non-typeable Haemophilus influenzae isolate, originally from an otitis media patent, was produced by on-filter growth. Extracellular matrix fraction was subjected to proteomic analysis via LC-MS/MS to identify proteins.
Article Reference Novel sialic acid transporter of Haemophilus influenzae.
Nontypeable Haemophilus influenzae is an opportunistic pathogen and a common cause of otitis media in children and of chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The lipooligosaccharides, a major component of the outer membrane of H. influenzae, play an important role in microbial virulence and pathogenicity. N-Acetylneuraminic acid (sialic acid) can be incorporated into the lipooligosaccharides as a terminal nonreducing sugar. Although much of the pathway of sialic acid incorporation into lipooligosaccharides is understood, the transporter responsible for N-acetylneuraminic acid uptake in H. influenzae has yet to be characterized. In this paper we demonstrate that this transporter is a novel sugar transporter of the tripartite ATP-independent periplasmic transporter family. In the absence of this transporter, H. influenzae cannot incorporate sialic acid into its lipooligosaccharides, making the organism unable to survive when exposed to human serum and causing reduced viability in biofilm growth.
Article Reference Antibiotic activity of telithromycin and comparators against bacterial pathogens isolated from 3,043 patients with acute exacerbation of chronic bronchitis.
Antimicrobial therapy is considered an important component in the medical management of most patients with acute exacerbation of chronic bronchitis (AECB). The three predominant bacterial species isolated are nontypeable Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae. Staphylococcus aureus is also frequently isolated while atypical bacteria are thought to cause up to 10% of exacerbations. Antibacterial resistance is increasing worldwide and little surveillance data exist concerning pathogens isolated from patients with AECB.
Article Reference Haemophilus influenzae oral whole cell vaccination for preventing acute exacerbations of chronic bronchitis.
Acute bronchitis leading to ongoing exacerbations is a serious condition predisposed to by viruses or bacteria. It can be fatal. Antibiotic therapy has not been particularly useful in clearing bacteria such as nontypeable Haemophilus influenzae (NTHi) because they colonise the upper respiratory tract. An oral NTHi vaccine has been developed to protect against recurrent acute episodes in chronic bronchitis.
Article Reference Antibiotics in the treatment of acute exacerbations of chronic bronchitis.
The benefit of antimicrobial therapy for patients with an acute exacerbation of chronic bronchitis (AECB) remains controversial for two main reasons. First, the distal airways of patients with chronic bronchitis are persistently colonised, even during clinically stable periods, with the same bacteria that have been associated with AECB. Second, bacterial infection is only one of several causes of AECB. These factors have led to conflicting analyses on the role of bacterial agents and the response to antimicrobial therapy of patients with AECB. An episode of AECB is said to be present when a patient with chronic obstructive pulmonary disease (COPD) experiences some combination of increased dyspnoea, increased sputum volume, increased sputum purulence and worsening lung function. While the average COPD patient experiences 2 - 4 episodes of AECB per year, some patients, particularly those with more severe airway obstruction, are more susceptible to these attacks than others. Bacterial agents appear to be particularly associated with AECB in patients with low lung function and those with frequent episodes accompanied by purulent sputum. Non-typeable Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis account for up to 50% of episodes of AECB. Gram-negative bacilli are more likely to occur in patients with more severe lung disease. Antibiotics have been used to ameliorate AECB, to prevent AECB and to prevent the long-term loss of lung function that characterises COPD. Numerous prevention trials have been conducted with fairly consistent results; antibiotics do not lessen the number of episodes of AECB but do reduce the number of days lost from work. Most antibiotic trials have studied the impact of treatment on episodes of AECB and results have been inconsistent, largely due to patient selection and end point definition. In patients with severe airway obstruction, especially in the presence of purulent sputum, antibiotic therapy significantly shortens the duration of symptoms and can be cost-effective. Over the past 50 years, virtually all classes of antimicrobial agents have been studied in AECB. Important considerations include penetration into respiratory secretions, spectrum of activity and antimicrobial resistance. These factors limit the usefulness of drugs such as amoxicillin, erythromycin and trimethoprim-sulfamethoxazole. Extended-spectrum oral cephalosporins, newer macrolides and doxycycline have demonstrated efficacy in clinical trials. Amoxicillin-clavulanate and flouoroquinolones should generally be reserved for patients with more severe disease. A number of investigational agents, including ketolides and newer quinolones, hold promise for treatment of AECB.
Article Reference Nontypeable Haemophilus influenzae in the lower respiratory tract of patients with chronic bronchitis.
The frequency of colonization and intracellular localization of nontypeable Haemophilus influenzae (NTHi) in the lower respiratory tract was determined in healthy adults and in clinically stable and acutely ill chronic bronchitis (CB) patients. NTHi was recovered from bronchial wash or bronchial brush specimens in 6 of 23 (26%) stable CB patients and in 1 of 15 (7%) CB patients with a respiratory exacerbation. No NTHi (0 of 26) was recovered from lower tract specimens of healthy adults undergoing anesthesia for elective surgery. Molecular typing of NTHi strains revealed that five of nine patients with stable CB had different strains in upper respiratory tract and bronchial wash/brush specimens collected simultaneously. Four stable patients with CB had different strains recovered on repeat bronchoscopy. These results demonstrate the frequent colonization of the lower airways of stable CB patients with multiple strains of NTHi. Bronchial biopsies also were examined for intracellular NTHi by in situ hybridization and immunofluorescence microscopy. Intracellular NTHi were found in 0 of 7 healthy adults, 8 of 24 patients with clinically stable CB, and 13 of 15 acutely ill CB patients. This observation suggests a role for intracellular infection by NTHi in the pathogenesis of exacerbations of CB.
Article Reference Airway inflammation and etiology of acute exacerbations of chronic bronchitis.
The etiologic role of bacterial pathogens isolated from sputum culture in 40 to 50% of acute exacerbations of chronic bronchitis (AECB) is controversial. If bacterial pathogens cause these AECB, they should be associated with greater neutrophilic airway inflammation than pathogen-negative exacerbations.
Article Reference Haemophilus influenzae in chronic bronchitis.
Colonization of the adult respiratory tract with nontypable Haemophilus influenzae is a dynamic process with new strains being acquired and replacing old strains periodically. The organism is a common cause of exacerbations of chronic bronchitis based on 3 lines of evidence: quantitative culture of the lower airways obtained by protected specimen brush, antibiotic trials, and serological studies. Nontypable H. influenzae expresses multiple adhesin molecules that mediate adherence to the respiratory tract mucosa. Recent studies have established that the bacterium penetrates the mucosal surface and survives intracellularly and in the interstitium of the submucosa. The organism shows a remarkable degree of antigenic diversity on its surface, including phase variation of lipooligosaccharide, antigenic heterogeneity of surface proteins, point mutations in genes encoding surface proteins and horizontal transfer of genes. These strategies facilitate evasion of the human immune response. Substantial progress has been made in identifying vaccine antigens to prevent infections caused by nontypable H. influenzae.
Article Reference A P5 peptide that is homologous to peptide 10 of OprF from Pseudomonas aeruginosa enhances clearance of nontypeable Haemophilus influenzae from acutely infected rat lung in the absence of detectable peptide-specific antibody.
Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen associated with otitis media and the exacerbation of chronic bronchitis. This study reports the vaccine potential of three peptides representing conserved regions of the NTHi P5 outer membrane protein which have been fused to a promiscuous measles virus F protein T-cell eptitope (MVF). The peptides correspond to a region in surface loop one (MVF/L1A), the central region of loop four (MVF/L4), and a C-terminal region homologous to peptide 10 of OprF from Pseudomonas aeruginosa (MVF/H3). Immunization of rats with MVF/H3 was the most efficacious in significantly reducing the number of viable NTHi in both the broncho-alveolar lavage fluid (74%) and lung homogenates (70%), compared to control rats. Importantly, despite significantly increased rates of clearance, immunization with MVF/H3 elicited poor antibody responses, suggesting that cell-mediated rather than humoral responses play an important role in the enhanced clearance of NTHi in this model.
Article Reference A method for the purification and refolding of a recombinant form of the nontypeable Haemophilus influenzae P5 outer membrane protein fused to polyhistidine.
Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen, commonly associated with otitis media and exacerbations of chronic bronchitis. Studies concerning the pathogenesis of NTHi have proposed an important function for P5, an outer membrane protein believed to play a role in the initiation of infection by mediating adherence to respiratory mucin. P5 has also generated interest as a potential vaccine candidate. In a previous study, an NTHi library screen with antibodies raised against P5 purified from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the purified protein was contaminated with closely migrating proteins. Consequently, the aim of this study was to express P5 in a heterologous system to overcome potential contamination with NTHi proteins that may complicate analytical or vaccine studies. Recombinant P5, with an N terminal extension of 10 residues that included six histidines, was cloned and expressed in Escherichia coli. The rP5 was purified with the Talon metal affinity resin in a denatured form and then refolded by incorporation into mixed-detergent micelles of octylglucoside and SDS. Circular dichroism of the refolded rP5 demonstrated 55% beta-strand content, which is consistent with the beta-strand content of native P5 and the homologous E. coli protein OmpA.
Article Reference Human immune response to nontypeable Haemophilus influenzae in chronic bronchitis.
Nontypeable Haemophilus influenzae (NTHI) causes recurrent respiratory tract infections in patients with chronic bronchitis. To elucidate the human immune response to NTHI, sera from 2 patients with exacerbations of chronic bronchitis due to NTHI were characterized. Both patients developed new bactericidal antibodies following infection. Immunoblot assays with homologous strains revealed antibodies to many antigens, with minimal difference between pre- and postexacerbation sera. By contrast, whole cell radioimmunoprecipitation, which detects antibodies exclusively to epitopes exposed on the bacterial surface, revealed that both patients made new antibodies to a limited number of antigens following infection, including P2, the major outer membrane protein of NTHI. Adsorption experiments showed that strain-specific, surface-exposed epitopes on the P2 molecule are targets for bactericidal antibodies. These results indicate that new bactericidal antibodies following infection by NTHI recognize antigenically heterogeneous surface-exposed epitopes on P2 and other surface proteins of NTHI.
Article Reference A possible role for lysozyme in determining acute exacerbation in chronic bronchitis.
The aggregation of non-serotypable Haemophilus influenzae (NTHI) by whole saliva from patients with chronic obstructive lung disease (COLD) was investigated. Significant differences were observed between salivary aggregating activity of a control and COLD population (P < 0.001). Saliva from patients less prone to acute exacerbations had a greater capacity to aggregate bacteria compared with saliva from patients with a predilection to infection. The mechanism of saliva-mediated aggregation of NTHI was investigated and shown to be related to lysozyme content. Lysozyme activity in saliva was measured by the turbidimetric technique and results showed that patients with chronic bronchitis had increased levels of salivary lysozyme, with a subpopulation within the non-infection-prone group having greater amounts. A significant difference was observed in salivary lysozyme between controls and non-infection-prone (P < 0.005) and infection-prone (P < 0.05) patients, respectively: the non-infection-prone patients having significantly (P < 0.005) more than the infection-prone patients. There was significant correlation (r = 0.742, P < 0.001) between salivary aggregation of NTHI and lysozyme activity. Chromatographically purified human lysozyme had a similar aggregation profile to that of saliva. There was no difference in serum and saliva lactoferrin concentrations between groups, but there was a significant increase (P < 0.05) in serum lysozyme concentration in the non-infection-prone group. This study suggests that the level of salivary lysozyme derived from macrophages may play an important role in determining resistance or susceptibility to acute bronchitis.
Article Reference Adhesion of nontypeable Haemophilus influenzae from blood and sputum to human tracheobronchial mucins and lactoferrin.
Nontypeable Haemophilus influenzae strains are the most common pathogens encountered in patients with chronic bronchitis. These organisms chronically colonize the airways of patients and occasionally cause bacteremia. Nontypeable H. influenzae strains have been demonstrated microscopically to bind to mucus, but quantitative studies of adhesion have not been published to date. We have therefore developed a reproducible microtiter plate assay to study mucin binding and have examined the adhesion of sputum and blood strains of nontypeable H. influenzae. The assay is similar to that described for Pseudomonas aeruginosa (S. Vishwanath and R. Ramphal, Infect. Immun. 45:197-202, 1984), but notably 2% Tween 20 is used to desorb bacteria from the wells to quantitate bacterial binding. Using a standard strain, we have established that 1 h of incubation is optimum with an inoculum of < or = 5 x 10(8) CFU/ml. The standard strain binds to bronchitic and cystic fibrosis mucins equally well but binds less to bronchiectasis mucins. It does not bind to bovine serum albumin or fetuin. We have also examined the levels of adhesion of freshly isolated sputum and bacteremia strains and find very significant differences in adhesion. Blood strains bound six to seven times less than sputum strains ([13.8 +/- 7] x 10(2) per well versus [102 +/- 43] x 10(2); P < 0.001). Studies with adhesion to lactoferrin, another glycosylated protein, revealed variable binding of respiratory strains but marked binding of blood strains compared with mucin. An isogenic pair of respiratory and blood isolates was examined by electron microscopy but did not show surface differences. We speculate that bacteremic strains studied may have masked, lost, or downregulated adhesin production to allow them to escape from mucins or upregulated adhesins for lactoferrin to invade the bloodstream.
Article Reference Conservation of immune responses to proteins isolated by preparative polyacrylamide gel electrophoresis from the outer membrane of nontypeable Haemophilus influenzae.
Outer membrane proteins P2, P4, and P6 and two with molecular masses of 26 and 28 kDa have been purified from a strain of nontypeable Haemophilus influenzae by a preparative form of polyacrylamide gel electrophoresis (PAGE). Outer membrane protein P6, with a molecular mass of 16 kDa (determined by sodium dodecyl sulfate [SDS]-PAGE) was purified by both native PAGE and SDS-PAGE from three strains of nontypeable H. influenzae and one strain of type b H. influenzae. The same conditions were required for purification from each strain. The suitability of proteins isolated by these methods was assessed by studying the immune response of rats immunized with P6 in incomplete Freund's adjuvant into the Peyer's patches. P6 purified by either native PAGE or SDS-PAGE did not differ significantly from P6 purified by gel filtration and anion-exchange chromatography in the ability to enhance pulmonary clearance of live bacteria. This study also investigated the effects of SDS on P2 immunological responses in vivo and the effects of the reagents Zwittergent and sodium lauryl sarcosinate on outer membrane protein lymphocyte-proliferative responses in vitro. It was found that the presence of SDS in the immunization emulsion enhanced the antigen-specific cell-mediated response but suppressed the antigen-specific antibody responses. The presence of residual traces of Zwittergent in an outer membrane protein preparation inhibited antigen-specific cell-mediated proliferation, whereas extraction of outer membrane proteins with sodium lauryl sarcosinate did not inhibit antigen-specific proliferation. These results demonstrate that preparative PAGE is a suitable method for the purification of proteins from the outer membrane of H. influenzae required for investigation of their immunological significance as vaccine candidates and that traces of reagents used during protein purification may play an important role in determining the success of in vivo and in vitro studies.
Article Reference Effect of primary immunization on pulmonary clearance of nontypable Haemophilus influenzae.
Nontypable Haemophilus influenzae (NTHI) is being increasingly recognized as a cause of both adult pneumonia and acute infectious exacerbations in chronic bronchitis. We used a mouse model to study the immune enhancement of pulmonary clearance of NTHI after a primary immunization. BALB/c mice were immunized with whole NTHI either by intraperitoneal (i.p.) or intratracheal (i.t.) routes. There was 10-fold more NTHI-directed antibody detected in the serum of the i.p.-immunized mice than in the serum from the i.t.-immunized animals. Western blot analysis revealed that these antibodies were directed against both NTHI lipooligosaccharide and the various outer membrane proteins of NTHI. The development of NTHI-directed antibodies in serum was associated with significant enhancement of early pulmonary clearance of NTHI. Six hours after delivery of an endobronchial challenge with NTHI, the i.p.-immunized mice had cleared most of the organisms from their lungs, while the i.t.-immunized mice did not clear NTHI any more rapidly than did unimmunized mice. Serum from the i.p.-immunized mice caused more than 99% uptake of NTHI in an in vitro opsonophagocytic assay, while serum from i.t.-immunized mice stimulated little or no phagocytosis of this organism. Opsonophagocytosis of NTHI was obtained with bronchoalveolar lavage (BAL) fluid collected from i.p.-immunized mice 6 h after, but not before, an endobronchial challenge with NTHI. Intravenous injection of an opsonic IgG monoclonal antibody directed against NTHI lipooligosaccharide resulted in both the appearance of this antibody in the alveolar spaces of the unperturbed lung and enhanced pulmonary clearance of NTHI. These data indicate that the i.p. (systemic) route of immunization is more effective than the i.t. route in establishing pulmonary immunity to NTHI in this model system. Furthermore, immune enhancement of clearance of NTHI from the lungs after a primary immunization apparently results from the exudation of opsonic and bactericidal antibodies from the serum into the alveolae in response to the inflammatory challenge.
Article Reference Nontypable Haemophilus influenzae: a review of clinical aspects, surface antigens, and the human immune response to infection.
Nontypable Haemophilus influenzae has now become well established as an important pathogen in both adults and children. Recent work has identified clear distinctions between nontypable and type b strains of H. influenzae. These organisms affect different patient populations, cause different infections, present different surface antigens to the host, and are genetically different. The commonest clinical manifestation of infection due to nontypable H. influenzae in adults is lower respiratory tract infection, particularly in the elderly and in those with chronic bronchitis. The bacterium is a frequent cause of acute otitis media in children. The surface of nontypable H. influenzae is composed of outer-membrane proteins and lipooligosaccharide, and both of these demonstrate substantial antigenic heterogeneity, which can be used to serotype isolates. Some respiratory tract isolates are fimbriated, but the role of fimbriae in pathogenesis is unclear. Antibodies to outer-membrane proteins and lipooligosaccharide are present in human serum. Investigation of human immunity to infection is focusing on identification of those antigens to which protective antibody is directed.
Article Reference Immunoglobulin A from bronchopulmonary secretions blocks bactericidal and opsonizing effects of antibody to nontypable Haemophilus influenzae.
Patients with chronic bronchitis are colonized by and may develop acute bronchopulmonary infection due to nontypable Haemophilus influenzae (NTHI) despite the presence of bactericidal and opsonizing antibody to the infecting organism. To test the hypothesis that secretory immunoglobulin A (IgA) interferes with host defense mechanisms, we extracted secretory IgA from bronchopulmonary secretions of five patients with NTHI pneumonia. NTHI was incubated with IgA before or during incubation with each patient's own serum or normal human serum. IgA from four of these individuals blocked the bactericidal and opsonizing effects of normal human serum and/or their own serum against their own and/or other NTHI. IgA from bronchopulmonary secretions of patients not infected with NTHI or from the serum of a patient with an IgA myeloma had no such effect. Blocking appeared to result from a direct interaction between IgA and the bacteria. The presumed mechanism is an interaction with bacterial surface antigens, although it is not known whether this occurs at antigenic sites responsible for bactericidal and opsonizing activity or whether interaction with adjacent antigenic sites and subsequent steric interference is responsible. This blocking effect of IgA may be one mechanism that allows for the development of NTHI colonization or pneumonia in an individual who already has seemingly adequate antibody against the infecting organism.
Chronic Bronchitis
Article Reference Overlapping and complementary oxidative stress defense mechanisms in nontypeable Haemophilus influenzae.
The Gram-negative commensal bacterium nontypeable Haemophilus influenzae (NTHi) can cause respiratory tract diseases that include otitis media, sinusitis, exacerbations of chronic obstructive pulmonary disease and bronchitis. During colonization and infection, NTHi withstands oxidative stress generated by reactive oxygen species produced endogenously, by the host and by other co-pathogens and flora. These reactive oxygen species include superoxide, hydrogen peroxide (H2O2)and hydroxyl radicals. This killing is amplified by iron, via the Fenton reaction. We previously identified genes that encode proteins with putative roles in protection of the NTHi isolate, strain 86-028NP against oxidative stress. These include catalase (HktE), peroxiredoxin/glutaredoxin (PgdX) and a ferritin-like protein (Dps). Strains were generated with mutations in hktE, pgdX and dps. The hktE mutant and a pgdX-hktE double mutant were more sensitive to killing by H2O2, when compared to the parent. Conversely, the pgdX mutant was more resistant to H2O2 due to increased catalase activity. Supporting the role of killing via the Fenton reaction, binding of iron by Dps significantly mitigated the effect of H2O2-mediated killing. NTHi thus utilizes several effectors to resist oxidative stress and regulation of free iron is critical to this protection. These mechanisms will be important for successful colonization and infection by this opportunistic human pathogen.