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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.
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Keywords: chronic bronchitis; colonization; intracellular; NTHi
Bacterial infections are thought to be the leading cause of exacerbations in patients with chronic obstructive pulmonary disease (COPD) (1). Nontypeable Haemophilus influenzae (NTHi) has been identified as an important pulmonary bacterial pathogen in association with recurrent and persistent lower respiratory tract infections in patients with COPD (1). Although NTHi is frequently recovered from the upper airway of healthy individuals and patients with COPD, the frequency of lower respiratory tract colonization in healthy adults and patients with COPD has not been clearly defined (4). Persistence of NTHi in the lower respiratory tract has been suggested to be a major determinant of pulmonary morbidity in patients with COPD (11). However, few studies have used bronchoscopic sampling methods to study lower respiratory tract colonization both in healthy subjects and patients with COPD during clinically stable periods and during acute exacerbations. The goals of the present studies were to (1) determine the frequency of colonization of the lower airways of healthy adults and of adults with COPD during clinically stable periods and during acute exacerbations, (2) determine the frequency of colonization by multiple strains of NTHi, and (3) characterize the intracellular location of NTHi in bronchial biopsies in patients with COPD during clinically stable periods and during exacerbations. These studies were performed to help clarify the role of NTHi and suggest possible pathogenetic mechanisms involved in exacerbation of chronic bronchitis in persons with COPD.
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Patient Populations
Patients with clinically stable chronic bronchitis (CB) were asked to participate. The study was approved by the Institutional Review Board of the Baylor College of Medicine (Houston, TX) and written informed consent was obtained from all subjects before initiating these studies. Exclusion criteria are listed in the expanded METHODS section (see online data supplement).
Adult patients who were having elective surgical procedures under general anesthesia were recruited for the control group. Bronchoscopy was performed immediately after intubation and completed before the infusion of prophylactic antibiotics, if any were used.
CB patients with signs and symptoms of acute exacerbations, including increased dyspnea, increased cough, and increased sputum production, were enrolled if they required intubation and mechanical ventilation.
Bronchoscopy Procedure
Standard methods were used for bronchoscopy, protected specimen brush, minibronchoalveolar lavage, and biopsies.
Specimens
Sputum and throat swab specimens were collected and transported on ice to the laboratory within 4 h of collection for bacteriologic studies.
Quantification and Identification
Bacteria were quantitated on selective agar plates and identified by previously described methods (14).
Long PCR Ribotyping
Purified DNA from isolates of NTHi were subjected to long polymerase chain reaction (PCR) ribotyping by a modification of the method of Smith-Vaughan and coworkers (20, 21).
Typing by SDS-PAGE
Strains of NTHi were subjected to typing by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole bacterial cell lysates (22).
Isolates were assigned types on the basis of banding patterns in gels. The first isolate from each patient was assigned the letter A, with each subsequent isolate with a different banding pattern being assigned consecutive letters. The lettering system is based on typing within each individual patient. Thus, type A from one patient is not the same as type A from another patient.
In Situ Hybridization
In situ hybridization was performed according to the method of Forsgren and coworkers (23). Sections were probed with a digitonin-labeled nucleotide sequence that specifically recognizes H. influenzae 16S rRNA and counterstained with a fluorescein-labeled anti-digitonin monoclonal antibody (MAb). Sections were examined with a Bio-Rad (Hercules, CA) 1026 laser scanning confocal microscope (LSCM).
Fluorescent Antibody Analysis
Bronchial biopsies were immunolabeled for the presence of H. influenzae, using a murine MAb (3B9, IgG2b isotype) specific for the P6 protein of Haemophilus species (24). Tissue was counterstained with a fluorescein-labeled goat anti-murine IgG antibody. Sections obtained from intubated acutely ill patients with CB also were incubated with MAb T-15 (IgA antibody). MAb T-15 binds to the phosphorylcholine epitope found on H. influenzae lipo-oligosaccharide. Studies of a limited number of strains indicate that this epitope is not expressed on Haemophilus parainfluenzae lipo-oligosaccharide. Tissue was counterstained with a Texas Red-conjugated anti-mouse IgA antibody. Tissues from normal subjects and from patients with stable CB were not studied with antibody T-15. Tissues were examined with a Bio-Rad 1026 LSCM.
Statistical Analysis
Data were analyzed with the SPSS (Chicago, IL) for Windows software package. Proportions were analyzed by the 
2 or Fisher exact
test. Continuous variables were analyzed by the Student t test. Significant differences were defined as those for which the probability of occurrence was 5% or less.
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Bronchoscopy Findings in Patients with Stable CB
Twenty-three patients with stable chronic bronchitis and 26 adult control subjects were evaluated for colonization with NTHi, H. parainfluenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. The two groups differed in mean age, frequency of males, and number of Hispanic participants (Table 1). Recovery of NTHi from throat swab specimens was not significantly different between the two groups. However, NTHi was identified in 26% of the bronchial specimens from the patients with CB whereas no NTHi was recovered from the bronchial specimens of the control subjects (Table 2, p = 0.007). Sixteen of the patients with CB consented to a second bronchoscopy, which was performed a mean of 6.4 mo later (range, 4.2-9.8 mo), and NTHi was isolated from the lower respiratory tract with a frequency similar to that seen with the first bronchoscopy. Four of the five patients with CB who had bronchial specimens from the first bronchoscopy positive for NTHi also had NTHi isolated from bronchial specimens from the second bronchoscopy.
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Bronchial wash samples that grew NTHi ranged in quantity
from 1 × 101 to 3.8 × 103 CFU/ml. Seven positive samples had
between 101 and 102 CFU/ml. Three positive samples had
103 CFU/ml. Bronchial brush samples that grew NTHi ranged
in quantity between 101 and 102 CFU/ml. Four positive samples grew 101 CFU/ml and two grew 102 CFU/ml.
Haemophilus parainfluenzae was recovered from more than one-half of the throat swab specimens of control subjects (23 of 26 [88%]) and patients with stable CB (24 of 39 [62%] overall; 18 of 23 initial and 6 of 16 follow-up). Bronchial wash specimens were positive for H. parainfluenzae in 46% of specimens from patients with of stable CB (11 of 23 initial and 7 of 16 follow-up) compared with 0% in control subjects (p = 0.001, Fisher exact test).
Streptococcus pneumoniae and M. catarrhalis were infrequently recovered from sputum, throat swab, and bronchial wash or brush specimens from patients with stable CB and control subjects. Streptococcus pneumoniae was recovered from 15% (6 of 39) of the stable CB patient sputum or throat swab specimens (4 of 23 initial and 2 of 16 follow-up) and 4% (1 of 26) of those (throat swab only) from control subjects. Streptococcus pneumoniae was recovered from 18% (7 of 39) of stable CB patient bronchial wash or brush specimens (3 of 23 initial and 4 of 16 follow-up) and 7% (2 of 26) of those from control subjects. Moraxella catarrhalis was recovered from 13% (5 of 39) of the stable CB patient sputum and throat swab specimens (5 of 23 initial and 0 of 16 follow-up) and 12% (3 of 26) of those from control subjects. Moraxella catarrhalis was recovered from 5% (2 of 39) of the stable CB patient bronchial wash or brush specimens (2 of 23 initial and 0 of 16 follow-up) and 4% (1 of 26) of those from control subjects.
The upper respiratory tract was sampled on the same day (n = 32) or within 14 d (n = 7) of the bronchoscopy, and NTHi was recovered from both upper and lower respiratory sites from 13 bronchoscopies in 9 patients with CB (Table 3). A total of 37 isolates of NTHi from these patients were subjected to long PCR ribotyping and outer membrane protein typing by SDS-PAGE of whole bacterial cell lysates (Figure 1). In all cases, both methods yielded identical results. In five bronchoscopies, upper and lower respiratory tract strains were different. Four individuals had two bronchoscopies at least 4 mo apart. In all four instances, strains recovered from the second bronchoscopies were different from those recovered from the first, indicating that the patients were colonized by new strains.
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In Situ Hybridization and Fluorescent Antibody Studies of Bronchoscopic Biopsies from Normal Subjects and Patients with Stable CB
The microscopic examination of all biopsies was performed in a blinded fashion. Coded specimens from patients and normals were examined and the source of the sample was not revealed to the microscopist until after the biopsy results were reported.
In situ hybridization and fluorescent antibody analysis failed to reveal the presence of H. influenzae in the bronchial biopsies from normal patients (0 of 10 patients). Fluorescent antibody analysis using MAb 3B9 (reacts with a conformational epitope present on the highly conserved P6 protein of H. influenzae) revealed the presence of H. influenzae within airway epithelial cells in the bronchial biopsies from patients with stable CB (Figure 2B). This group of patients was also studied by in situ hybridization to detect the presence of H. influenzae intracellularly (Figure 2A). The results of both analyses indicated that 8 of 24 patients with stable CB had evidence of intracellular H. influenzae. Subsequent analysis showed that three of the eight patients with stable chronic bronchitis who were positive by these analyses had NTHi cultured from their bronchial secretions. Five of the 16 patients with negative biopsies had NTHi cultured from their bronchial secretions.
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Intubated, Acutely Ill Patients with CB
Fifteen acutely ill patients with CB, requiring intubation for respiratory failure, had bronchoscopy performed within 18 h of admission and after at least one dose of antibiotic had been given. Additional clinical information about these subjects is provided in the online data supplement. NTHi was recovered from one and H. parainfluenzae from two of the bronchial wash specimens. No M. catarrhalis or S. pneumoniae was isolated. Bronchial biopsies were collected and examined for intracellular NTHi. Figure 3A-3D shows a typical LSCM micrograph from a biopsy from such a patient. As can be seen, Haemophilus (identified by MAb 3B9) are present within airway epithelial cells. In addition, the samples were reacted with monoclonal antibody T-15 (recognizes the phosphorylcholine epitope on H. influenzae lipo-oligosaccharide). This structure has been shown to be the ligand for the entry of NTHi into human airway epithelial cells via the human platelet-activating factor receptor (PAF-R) (25). It is interesting to note that every organism that was found to react with MAb 3B9 also reacted with MAb T-15. This is consistent with the data indicating that this phase-varying epitope must be present for intracellular entry. Intracellular phosphorylcholine-positive NTHi was found in 13 of 15 (87%) of these acutely ill patients (Table 4). The combination of studies with MAbs 3B9 and T-15 suggests that the intracellular Haemophilus are H. influenzae.
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These results demonstrate that NTHi is frequently found in the lower airways of patients with CB when clinically stable and acutely ill. NTHi was recovered from the lower airways of 25-50% of stable and 7% of acutely ill CB patients. The lower frequency of recovery in the acutely ill patients is most likely due to the universal administration of parenteral antibiotics in these intubated patients before obtaining the lower respiratory tract cultures. In addition, NTHi could be demonstrated intracellularly in 33% of patients with stable CB and 87% of acutely ill patients. In contrast, we did not find NTHi in the lower respiratory tract of healthy adults.
Other studies have demonstrated colonization of the lower airways with NTHi and other pathogenic bacteria in patients with stable COPD (7, 9, 10, 26). In patients with CB, NTHi appears to be associated with recurrent and/or persistent infections of the lower respiratory tract. NTHi appears to be able to persist in the lower respiratory tract for months and can be isolated even after or during antimicrobial treatment (27, 28). The initial step in NTHi colonization of the lower respiratory tract is adherence to the surface of epithelial cells (29). Several groups have demonstrated that NTHi may reside between the epithelial cells and in the subepithelial layers of the lower respiratory tract (30, 31).
Tissue culture studies, using primary human airway epithelial cells grown at an air-fluid interface and simian virus 40 (SV-40)-transformed bronchial epithelial cells, indicate that NTHi can enter and survive within airway epithelial cells (24, 25, 32). These studies suggest at least two distinct mechanisms for entry, both of which are dependent on inositol triphosphate kinase (IP-3 kinase) signaling. The most efficient mechanism for entry appears to be through the interaction of the phosphorylcholine residue on the NTHi lipo-oligosaccharide with the human PAF-R. This results in a signal cascade through IP-3 kinase that results in the release of intracellular Ca2+ (24, 33). Blockade of PAF-R with specific inhibitors reduces invasion by more than 90%. Macropinocytosis is a second mechanism for entry. It is not modified by inhibition of PAF-R and also appears to signal through IP-3 kinase. In our studies, we have shown that NTHi can be found within or between cells in bronchial biopsies from acutely ill, intubated CB patients as well as patients with stable CB who are culture positive. Although the resolution of the microscopy does not allow definition of the exact location of NTHi in these patient samples, the in vitro data cited above suggest that these bacteria most probably are intracellular. Several of the patients with stable CB had NTHi isolated from the lower bronchial tree but did not have these organisms detected in their biopsy specimens. Although failure to detect intracellular NTHi in these colonized subjects could represent sampling error, another possibility is that the colonizing NTHi did not express the phosphorylcholine residue on the lipo-oligosaccharide. NTHi has been shown to undergo phase variation in its decoration of the lipo-oligosaccharide with phosphorylcholine (34). We did not specifically evaluate phosphorylcholine expression in the NTHi isolates collected during the study.
Colonization of the lower respiratory tract with NTHi may
lead to increased inflammation and lung damage. NTHi induces increased proinflammatory cytokine (e.g., interleukin 8 [IL-8], IL-6, and tumor necrosis factor 
[TNF-]) expression
in an in vitro model of human respiratory epithelial cells (35).
In clinical studies, increased concentrations of sputum transferrin levels in CB patients with NTHi were seen compared
with control subjects or subjects with asthma (35). Secretory
IgA levels were higher in the sputum sol phase from CB patients with NTHi compared with noncolonized patients with
CB. Bacteria-specific factors may also play a role. When NTHi
strains from persistently colonized patients with CB (six of six
monthly cultures positive for NTHi) were compared with
those from intermittently colonized patients with CB (one of
six monthly cultures positive), persisting strains induced less
IL-6 and IL-8 than nonpersisting strains (in a lung epithelial cell model system) (36). The importance of inflammatory mediators in patient outcome is suggested by the finding of increased levels of inflammatory markers (myeloperoxidase and
TNF-) in the sputum sol phase of CB patients chronically infected with NTHi and having irreversible airway obstruction
compared with patients with CB who were nonobstructed but
infected with NTHi (37). However, additional studies are
needed to confirm the importance of lower respiratory tract
colonization as a stimulus for inflammation and to determine
the nature of the host-bacteria interaction during an exacerbation (38).
Several studies have subjected multiple isolates of NTHi from the same sample to molecular typing (39, 40). These studies have revealed that the respiratory tract harbors multiple strains of NTHi in several clinical settings, including cystic fibrosis, CB, and Aboriginal children with a high incidence of otitis media. The present study is the first to characterize isolates from upper and lower respiratory sites simultaneously. Typing of strains of NTHi recovered from various sites in the respiratory tract revealed that colonization by multiple strains simultaneously was common in the setting of CB (Figures 1 and 2). This observation has several important implications. First, understanding the dynamics of colonization of the human respiratory tract is critical if strategies to prevent infection and/or colonization are to be developed. Second, these observations further suggest that results of sputum cultures may not reliably reflect the bacteriology in the lower respiratory tract, because in several instances the isolate recovered from expectorated sputum was different from the isolate recovered from bronchial wash or bronchial brush. Finally, an awareness that a strain of NTHi with a different antimicrobial susceptibility pattern compared with isolates in expectorated sputum has important implications for the clinician with regard to rational choice of antibiotic therapy.
There are certain potential limitations to the results of our study. The demographics of the control group were different from the patients with stable CB. Although sex, age, and ethnicity are not known to be risk factors for NTHi colonization, these could be confounding factors for our observed differences in NTHi colonization of the lower respiratory tract. Second, the acutely ill patients with CB had received antibiotics, limiting identification of any quantitative differences in NTHi recovered from the bronchial wash specimens. Third, sampling variability could alter the frequency and quantity of NTHi isolates from both upper and lower respiratory tract specimens. Last, the quality of the sputum samples was not rigorously defined to exclude contamination with saliva. However, our recovery rates of NTHi are similar to those in other published studies.
The results of our studies demonstrate a dissociation between sputum and lower respiratory cultures for NTHi. This could be a result of the number of colonies examined for strain characterization or reflect phenotypic differences in NTHi strains recovered from upper versus lower airways. Second, the number of NTHi strains varied over time and even at different sites at the same time. Third, the demonstration of intracellular NTHi in lower respiratory tract cells from patients with CB supports the finding from in vitro cell culture systems. Future studies will examine the quantity of inflammatory mediators from the lower respiratory tract of these patients with CB to see whether they correlate with levels found in sputum samples obtained from persistently colonized samples from NTHi-infected patients with CB.
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Correspondence and requests for reprints should be addressed to S. B. Greenberg, M.D., Department of Medicine, Baylor College of Medicine, Houston, TX 77030. E-mail: stepheng{at}bcm.tmc.edu
(Received in original form April 20, 2001 and accepted in revised form August 16, 2001).
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org
Acknowledgments: We thank Terry O'Neill, Nerisa Kershaw, Margaret Ketterer, Melvin Sunshine, and Jian Q. Shao for help in the performance of clinical and laboratory studies, Eula Landry for help in manuscript preparation, and Dr. Robert B. Couch for critical review of the data presented herein.
Supported by Public Health Service contract N01AI 65298 and grants AI 19641 and AI 24616 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health.
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  S. J. Moghaddam, H. Li, S.-N. Cho, M. K. Dishop, I. I. Wistuba, L. Ji, J. M. Kurie, B. F. Dickey, and F. J. DeMayo Promotion of Lung Carcinogenesis by Chronic Obstructive Pulmonary Disease-Like Airway Inflammation in a K-ras-Induced Mouse Model Am. J. Respir. Cell Mol. Biol., April 1, 2009; 40(4): 443 - 453. [Abstract] [Full Text] [PDF]
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 S. Sethi and T. F. Murphy Infection in the Pathogenesis and Course of Chronic Obstructive Pulmonary Disease N. Engl. J. Med., November 27, 2008; 359(22): 2355 - 2365. [Full Text] [PDF]
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S. J. Moghaddam, C. G. Clement, M. M. De la Garza, X. Zou, E. L. Travis, H. W. J. Young, C. M. Evans, M. J. Tuvim, and B. F. Dickey Haemophilus influenzae Lysate Induces Aspects of the Chronic Obstructive Pulmonary Disease Phenotype Am. J. Respir. Cell Mol. Biol., June 1, 2008; 38(6): 629 - 638. [Abstract] [Full Text] [PDF]
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 F. J. Martinez Pathogen-directed Therapy in Acute Exacerbations of Chronic Obstructive Pulmonary Disease Proceedings of the ATS, December 1, 2007; 4(8): 647 - 658. [Abstract] [Full Text] [PDF]
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 M. Miravitlles Review: Do we need new antibiotics for treating exacerbations of COPD? Therapeutic Advances in Respiratory Disease, October 1, 2007; 1(1): 61 - 76. [Abstract] [PDF]
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A. Anzueto, S. Sethi, and F. J. Martinez Exacerbations of Chronic Obstructive Pulmonary Disease Proceedings of the ATS, October 1, 2007; 4(7): 554 - 564. [Abstract] [Full Text] [PDF]
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 U. S. Sajjan, Y. Jia, D. C. Newcomb, J. K. Bentley, N. W. Lukacs, J. J. LiPuma, and M. B. Hershenson H. influenzae potentiates airway epithelial cell responses to rhinovirus by increasing ICAM-1 and TLR3 expression FASEB J, October 1, 2006; 20(12): 2121 - 2123. [Abstract] [Full Text] [PDF]
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D. C. Look, C. L. Chin, L. J. Manzel, E. E. Lehman, A. L. Humlicek, L. Shi, T. D. Starner, G. M. Denning, T. F. Murphy, and S. Sethi Modulation of Airway Inflammation by Haemophilus influenzae Isolates Associated with Chronic Obstructive Pulmonary Disease Exacerbation Proceedings of the ATS, August 1, 2006; 3(6): 482 - 483. [Full Text] [PDF]
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 M. M. Fernaays, A. J. Lesse, S. Sethi, X. Cai, and T. F. Murphy Differential Genome Contents of Nontypeable Haemophilus influenzae Strains from Adults with Chronic Obstructive Pulmonary Disease. Infect. Immun., June 1, 2006; 74(6): 3366 - 3374. [Abstract] [Full Text] [PDF]
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 A. Papi, C. M. Bellettato, F. Braccioni, M. Romagnoli, P. Casolari, G. Caramori, L. M. Fabbri, and S. L. Johnston Infections and Airway Inflammation in Chronic Obstructive Pulmonary Disease Severe Exacerbations Am. J. Respir. Crit. Care Med., May 15, 2006; 173(10): 1114 - 1121. [Abstract] [Full Text] [PDF]
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S. West-Barnette, A. Rockel, and W. E. Swords Biofilm Growth Increases Phosphorylcholine Content and Decreases Potency of Nontypeable Haemophilus influenzae Endotoxins Infect. Immun., March 1, 2006; 74(3): 1828 - 1836. [Abstract] [Full Text] [PDF]
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 E Sapey and R A Stockley COPD exacerbations {middle dot} 2: Aetiology. Thorax, March 1, 2006; 61(3): 250 - 258. [Abstract] [Full Text] [PDF]
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 G. B. Toews Impact of bacterial infections on airway diseases Eur. Respir. Rev., December 1, 2005; 14(95): 62 - 68. [Abstract] [Full Text] [PDF]
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R. Garcia-Medina, W. M. Dunne, P. K. Singh, and S. L. Brody Pseudomonas aeruginosa Acquires Biofilm-Like Properties within Airway Epithelial Cells Infect. Immun., December 1, 2005; 73(12): 8298 - 8305. [Abstract] [Full Text] [PDF]
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D. M. Kelly, C. M. Greene, G. Meachery, M. O'Mahony, P. M. Gallagher, C. C. Taggart, S. J. O'Neill, and N. G. McElvaney Endotoxin Up-regulates Interleukin-18: Potential Role for Gram-Negative Colonization in Sarcoidosis Am. J. Respir. Crit. Care Med., November 15, 2005; 172(10): 1299 - 1307. [Abstract] [Full Text] [PDF]
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 C. W. Wieland, S. Florquin, N. A. Maris, K. Hoebe, B. Beutler, K. Takeda, S. Akira, and T. van der Poll The MyD88-Dependent, but Not the MyD88-Independent, Pathway of TLR4 Signaling Is Important in Clearing Nontypeable Haemophilus influenzae from the Mouse Lung J. Immunol., November 1, 2005; 175(9): 6042 - 6049. [Abstract] [Full Text] [PDF]
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S. L. Johnston Overview of Virus-induced Airway Disease Proceedings of the ATS, August 1, 2005; 2(2): 150 - 156. [Abstract] [Full Text] [PDF]
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C. L. Chin, L. J. Manzel, E. E. Lehman, A. L. Humlicek, L. Shi, T. D. Starner, G. M. Denning, T. F. Murphy, S. Sethi, and D. C. Look Haemophilus influenzae from Patients with Chronic Obstructive Pulmonary Disease Exacerbation Induce More Inflammation than Colonizers Am. J. Respir. Crit. Care Med., July 1, 2005; 172(1): 85 - 91. [Abstract] [Full Text] [PDF]
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 A. Rosell, E. Monso, N. Soler, F. Torres, J. Angrill, G. Riise, R. Zalacain, J. Morera, and A. Torres Microbiologic Determinants of Exacerbation in Chronic Obstructive Pulmonary Disease Arch Intern Med, April 25, 2005; 165(8): 891 - 897. [Abstract] [Full Text] [PDF]
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T. F. Murphy, A. L. Brauer, A. T. Schiffmacher, and S. Sethi Persistent Colonization by Haemophilus influenzae in Chronic Obstructive Pulmonary Disease Am. J. Respir. Crit. Care Med., August 1, 2004; 170(3): 266 - 272. [Abstract] [Full Text] [PDF]
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 H. P. Jia, J. N. Kline, A. Penisten, M. A. Apicella, T. L. Gioannini, J. Weiss, and P. B. McCray Jr. Endotoxin responsiveness of human airway epithelia is limited by low expression of MD-2 Am J Physiol Lung Cell Mol Physiol, August 1, 2004; 287(2): L428 - L437. [Abstract] [Full Text] [PDF]
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Y. Qiu, J. Zhu, V. Bandi, R. L. Atmar, K. Hattotuwa, K. K. Guntupalli, and P. K. Jeffery Biopsy Neutrophilia, Neutrophil Chemokine and Receptor Gene Expression in Severe Exacerbations of Chronic Obstructive Pulmonary Disease Am. J. Respir. Crit. Care Med., October 15, 2003; 168(8): 968 - 975. [Abstract] [Full Text] [PDF]
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 W. E. Swords, P. A. Jones, and M. A. Apicella Review: The lipo-oligosaccharides of Haemophilus influenzae: an interesting array of characters Innate Immunity, June 1, 2003; 9(3): 131 - 144. [Abstract] [PDF]
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T. F. Murphy Immunity to Nontypeable Haemophilus influenzae: Elucidating Protective Responses Am. J. Respir. Crit. Care Med., February 15, 2003; 167(4): 486 - 487. [Full Text] [PDF]
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P. T. King, P. E. Hutchinson, P. D. Johnson, P. W. Holmes, N. J. Freezer, and S. R. Holdsworth Adaptive Immunity to Nontypeable Haemophilus influenzae Am. J. Respir. Crit. Care Med., February 15, 2003; 167(4): 587 - 592. [Abstract] [Full Text] [PDF]
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M. J. Tobin Compliance (COMmunicate PLease wIth Less Abbreviations, Noun Clusters, and Exclusiveness) Am. J. Respir. Crit. Care Med., December 15, 2002; 166(12): 1534 - 1536. [Full Text] [PDF]
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 M. Miravitlles Exacerbations of chronic obstructive pulmonary disease: when are bacteria important? Eur. Respir. J., July 1, 2002; 20(36_suppl): 9S - 19s. [Abstract] [Full Text] [PDF]
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M. J. TOBIN Critical Care Medicine in AJRCCM 2001 Am. J. Respir. Crit. Care Med., March 1, 2002; 165(5): 565 - 583. [Full Text] [PDF]
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M. J. TOBIN Chronic Obstructive Pulmonary Disease, Pollution, Pulmonary Vascular Disease, Transplantation, Pleural Disease, and Lung Cancer in AJRCCM 2001 Am. J. Respir. Crit. Care Med., March 1, 2002; 165(5): 642 - 662. [Full Text] [PDF]
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