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T F Murphy

Publications and source records attributed to T F Murphy.

At least 37 records · Page 2Linked to original sources
PubMed1999

Immunization of mice with P6 of nontypeable Haemophilus influenzae: kinetics of the antibody response and IgG subclasses.

The kinetics of the anti-P6 antibody response was characterized in three strains of mice of different haplotypes (Balb/c; H-2d, C3H/H; H-2k, SJL/J; H-2s). Anti-P6 antibodies were measured on a weekly basis by enzyme-linked immunosorbent assay (ELISA). The primary response peaked 2 or 3 weeks after the initial injection with 40 microg of purified P6. The response remained at a plateau for 8-10 weeks. A maximum titer of 1:1,638,400 was attained and then steadily declined. To study the ability of P6 to generate a recall response, we opted to boost the vaccinated mice with a known subimmunogenic dose of live nontypeable Haemophilus influenzae (NTHI) bacteria. After the anti-P6 antibody titers in the primed animals had stayed at baseline levels for 2 weeks, the mice were injected intraperitonealy with 10(8) cfu of NTHI in sterile saline. This challenge with live NTHI bacteria induced a very rapid and strong secondary antibody response in all mice. Finally, we demonstrated that these murine anti-P6 sera were 100% bactericidal against three strains of NTHI when tested in a complement dependant bactericidal assay.

Animals↗
PubMed1999

Simultaneous respiratory tract colonization by multiple strains of nontypeable haemophilus influenzae in chronic obstructive pulmonary disease: implications for antibiotic therapy.

Nontypeable Haemophilus influenzae often causes exacerbations of chronic obstructive pulmonary disease (COPD), and these exacerbations are frequently treated with oral antibiotics. The goals of this study were to determine the frequency of the simultaneous presence of multiple strains of H. influenzae in sputum and to measure the MICs of antibiotics for the isolates. In a prospective study, adults with COPD were seen monthly. Sputum cultures were obtained, and individual colonies were subjected to genomic DNA typing and MIC determinations. Multiple strains of H. influenzae were present simultaneously in the sputum of 26.3% of adults with COPD. In 64.5% of these, MICs of >/=1 antibiotic varied by >/=4-fold among the strains. Therefore, multiple strains of H. influenzae are frequently present simultaneously in the sputum of adults with COPD, and the antimicrobial susceptibility of different strains in the same sputum sometimes differs.

Aged↗
PubMed1999

Purification and characterization of outer membrane protein P6, a vaccine antigen of non-typeable Haemophilus influenzae.

Outer membrane protein P6 is a promising vaccine antigen with potential to prevent infections caused by non-typeable Haemophilus influenzae. A convenient and reliable method for the purification of P6 and an assessment of the purity, yield, protein structure, antigenicity and immunogenicity of the purified protein are described. The method begins with intact H. influenzae and utilizes a series of incubations and centrifugations using a single buffer to remove all cell components with the exception of the peptidoglycan to which the P6 is associated. P6 is dissociated from the complex with heat and the insoluble peptidoglycan is removed by centrifugation. The procedure yields highly purified P6. Contamination with lipooligosaccharide is less than 0.025 endotoxin U per microgr P6. The yield of P6 is approximately 2 mg of P6 per l H. influenzae culture. The purified P6 retains both the secondary and tertiary structure as measured by circular dichroism and analysis with monoclonal antibodies. The purified P6 is immunogenic in animals. A convenient method for purifying P6 which retains antigenicity and immunogenicity will be an important tool for future studies of the vaccine potential of P6.

Adult↗
PubMed1999

Analysis of antigenic structure and human immune response to outer membrane protein CD of Moraxella catarrhalis.

Moraxella catarrhalis is an important cause of otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease (COPD). Outer membrane protein CD (OMP CD) is a 45-kDa protein which is a potential vaccine antigen to prevent infections caused by M. catarrhalis. Eight monoclonal antibodies were used to study the antigenic structure of the OMP CD molecule by assaying recombinant peptides corresponding to the sequence of the protein. This approach identified two surface-exposed epitopes, including one near the amino terminus (amino acids 25 to 44) and one in the central region of the molecule (amino acids 261 to 331). Assays with serum and sputum supernatants of adults with COPD revealed variable levels of antibodies to OMP CD among individuals. To determine which portions of the OMP CD molecule were recognized by human antibodies, three human serum samples were studied with six recombinant peptides which span the sequence of OMP CD. All three sera contained immunoglobulin G antibodies which recognized exclusively the peptide corresponding to amino acids 203 to 260 by immunoblot assay. Adsorption experiments with whole bacteria established that some of the human antibodies are directed at surface-exposed epitopes on OMP CD. We conclude that OMP CD is a highly conserved molecule which contains at least two separate epitopes which are exposed on the bacterial surface. While individual adults with COPD show variability in the immune response to OMP CD, a specific region of the OMP CD molecule (amino acids 203 to 260) is important as a target of the human immune response.

Adult↗
PubMed1998

Human milk lactoferrin inactivates two putative colonization factors expressed by Haemophilus influenzae.

Haemophilus influenzae is a major cause of otitis media and other respiratory tract disease in children. The pathogenesis of disease begins with colonization of the upper respiratory mucosa, a process that involves evasion of local immune mechanisms and adherence to epithelial cells. Several studies have demonstrated that human milk is protective against H. influenzae colonization and disease. In the present study, we examined the effect of human milk on the H. influenzae IgA1 protease and Hap adhesin, two autotransported proteins that are presumed to facilitate colonization. Our results demonstrated that human milk lactoferrin efficiently extracted the IgA1 protease preprotein from the bacterial outer membrane. In addition, lactoferrin specifically degraded the Hap adhesin and abolished Hap-mediated adherence. Extraction of IgA1 protease and degradation of Hap were localized to the N-lobe of the bilobed lactoferrin molecule and were inhibited by serine protease inhibitors, suggesting that the lactoferrin N-lobe may contain serine protease activity. Additional experiments revealed no effect of lactoferrin on the H. influenzae P2, P5, and P6 outer-membrane proteins, which are distinguished from IgA1 protease and Hap by the lack of an N-terminal passenger domain or an extracellular linker region. These results suggest that human milk lactoferrin may attenuate the pathogenic potential of H. influenzae by selectively inactivating IgA1 protease and Hap, thereby interfering with colonization. Future studies should examine the therapeutic potential of lactoferrin, perhaps as a supplement in infant formulas.

Adhesins, Bacterial↗
PubMed1998

Serum antibodies specific to CD outer membrane protein of Moraxella catarrhalis, P6 outer membrane protein of non-typeable Haemophilus influenzae and capsular polysaccharides of Streptococcus pneumoniae in children with otitis media with effusion.

We measured the levels of serum IgG antibodies to CD outer membrane protein of Moraxella catarrhalis, P6 outer membrane protein of non-typeable Haemophilus influenzae and capsular polysaccharides of Streptococcus pneumoniae in 168 children with otitis media with effusion (OME) who were followed prospectively, using ELISA. Serum IgG antibodies to CD, P6 and pneumococcal capsular polysaccharides were detected in all samples. The anti-pneumococcal polysaccharides antibody level was highest, followed by the anti-P6 antibody level and anti-CD antibody was lowest (median:interquartile ranges were 45.9:19.1-100 microg/ml, 15.6:9.70-23.2 microg/ml and 1.06:0.73-1.87 microg/ml, respectively). In children aged 0-6 years, there were positive correlations among the antibody levels (anti-CD vs anti-P6, r=0.325, p <0.001; anti-CD vs anti-polysaccharide, r=0.397, p <0.0001; anti-P6 vs anti-polysaccharide, r=0.175, p=0.057). However, no relationship was seen in children aged 7-15 years. Children were classified according to severity of OME during the 1-year follow-up. In children aged 0-6 years, the severity of OME correlated inversely with the levels of anti-CD antibody (r=-.23, p=0.012), of anti-P6 antibody (r=-0.292, p=0.0015), and of anti-pneumococcal polysaccharides antibody (r=-0.25, p=0.0064). However, no correlation was found between antibody levels and severity of OME in children aged 7-15 years. These data suggest that persistence and/or recurrence of OME may be due to an insufficient serum antibody response to middle ear pathogens in young children.

Adolescent↗
PubMed1998

Enhancement of pulmonary clearance of Moraxella (Branhamella) catarrhalis following immunization with outer membrane protein CD in a mouse model.

Moraxella (Branhamella) catarrhalis is an important human respiratory tract pathogen. Outer membrane protein (OMP) CD is highly conserved among strains and has characteristics that indicate it may be an effective vaccine antigen. This study investigated the effect of immunization with OMP CD on pulmonary clearance following intratracheal challenge of mice with M. catarrhalis. Two routes of immunization were studied: mucosal immunization (intra-Peyer's patch followed by intratracheal boost) and intramuscular immunization with OMP CD. Both resulted in enhanced pulmonary clearance of M. catarrhalis compared with sham-immunized controls. Immunization with OMP CD induced specific antibodies in serum and bronchoalveolar lavage fluid and induced a specific lymphocyte proliferative response in T cells from mesenteric lymph nodes from mice mucosally immunized with OMP CD. On the basis of these results, OMP CD should undergo continued testing to determine whether it will induce a protective immune response in humans.

Animals↗
PubMed1998

Comparison between cefprozil and penicillin to eradicate pharyngeal colonization of group A beta-hemolytic streptococci.

BACKGROUND: Our objective was to perform a prospective, randomized, double blinded study of cefprozil and penicillin therapy to eradicate group A beta-hemolytic streptococci (GABHS) in children who were bacteriologic failures after receiving a standard 10-day course of penicillin treatment for GABHS pharyngitis. METHODS: Children and adolescents 2 to 18 years of age were eligible for the study. From 3 to 7 days after completing oral penicillin therapy for pharyngitis caused by GABHS, the study was explained, informed consent was obtained, a history and physical examination were completed and a throat culture was performed. Children with throat cultures positive for GABHS were randomized to receive either cefprozil or penicillin for 10 days. Children who were bacteriologic failures after administration of the first study drug were crossed over to receive the alternate drug. RESULTS: Of 180 enrolled children 66 (37%) had throat cultures positive for GABHS. Seventeen were excluded from the study, leaving 49 who completed the protocol. Of the 49 participants 26 received cefprozil initially whereas 23 received penicillin. GABHS were eradicated from the pharynx of 73% of children who received cefprozil as the first antibiotic compared with 39% of penicillin recipients (chi square, 5.748, 0.01 < P < 0.025). After crossover of failures, the final efficacy rate for cefprozil was 65% compared with 36.7% for penicillin (chi square, 5.523, 0.01 < P < 0.025). CONCLUSIONS: Cefprozil was more effective than penicillin in treating children who were bacteriologic failures after a standard 10-day course of oral penicillin.

Adolescent↗
PubMed1998

Outer-membrane antigen expression by Moraxella (Branhamella) catarrhalis influences pulmonary clearance.

Moraxella (Branhamella) catarrhalis is a common respiratory tract pathogen in man. The bacterium shows a strong tendency to form aggregates in vitro. A variant strain of M. catarrhalis that showed a reduced tendency to form aggregates was selected by successive in-vitro passage in broth culture from which aggregates had settled. The non-clumping variant strain showed alteration in expression of outer-membrane antigens, including the HMW-OMP, an outer-membrane protein of c. 200 kDa, outer-membrane protein CD and lipo-oligosaccharide. A mouse model for pulmonary challenge with M. catarrhalis revealed significant differences in the rate of clearance of the isogenic variant strains from the lung. The parent strain caused enhanced recruitment of neutrophils to the lung and more rapid clearance of bacteria from the lungs in comparison to the non-clumping variant. It is concluded that alteration of expression of surface molecules by M. catarrhalis has a significant impact in an in-vivo model of pulmonary clearance.

Adult↗
PubMed1997

Mechanisms of recurrent otitis media: importance of the immune response to bacterial surface antigens.

Otitis-prone children experience recurrent episodes of otitis media due to nontypeable H. influenzae (NTHI). A protective immune response occurs following infection, but this immune response is specific for the infecting strain, leaving the child susceptible to infection by other strains of NTHI. Little is known about the mechanism by which a strain-specific antibody response occurs to nonencapsulated bacteria. To explore the mechanism by which this strain-specific response occurs, animals were inoculated with whole bacterial cells and the antibody response was studied. The antibody response was predominantly directed to a highly strain-specific, immunodominant surface loop on the major outer membrane protein. This exquisitely restricted immune response leaves the host susceptible to recurrent infections by many strains of NTHI. The ability of the bacterium to direct the host to make a strain-specific antibody response has important implications in understanding the immune response to otitis media due to NTHI and in designing strategies for vaccine development.

Antigens, Bacterial↗
PubMed1997

Analysis of isolates recovered from multiple sites of the nasopharynx of children colonized by nontypeable Haemophilus influenzae.

To determine whether the nasopharynx of children is colonized by a single or multiple strains of nontypeable Haemophilus influenzae, cultures were obtained from six nasopharyngeal sites in five children. For each child, all isolates yielded identical polymerase chain reaction fingerprints. The results indicate that these children were colonized in the nasopharynx with a single strain of nontypeable Haemophilus influenzae at one time.

Child, Preschool↗
PubMed1997

Middle ear mucin glycoprotein: purification and interaction with nontypable Haemophilus influenzae and Moraxella catarrhalis.

Nontypable Haemophilus influenzae and Moraxella catarrhalis are important pathogens in children and adults. The mechanisms of their adherence to the epithelial cell surface and colonization are not clear. For the pathogen to adhere to the epithelial cell, it must first attach to and penetrate the mucus barrier. Mucin glycoproteins of the mucus layer generally are thought to be involved in bacterial attachment. To understand the precise mechanisms of middle ear mucin-bacterial interactions, we used an overlay binding assay with a highly purified middle ear mucin and outer membrane proteins of both nontypable H. influenzae and M. catarrhalis. Outer membrane proteins P2 and P5 were identified as the major components that medicate the binding between nontypable H. influenzae and human middle ear mucin. Moreover, the 57 kDa protein, CD, of the outer membrane protein of M. catarrhalis was found to be the only protein binding human middle ear mucin. Finally, it appears that a protein-oligosaccharide interaction is responsible for binding because asialo-mucin does not bind to either of the bacteria. Knowledge of the specific bacterial-mucin interaction may provide an understanding of the bacterial-epithelial cell colonization. Conversely, comprehension of this interaction between bacteria and purified mucin may be a strategy to prevent colonization of potential pathogens that cause otitis media and sinusitis in children.

Amino Acids↗
PubMed1997

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.

Antibodies, Bacterial↗
PubMed1997

Importance of an immunodominant surface-exposed loop on outer membrane protein P2 of nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHI) frequently causes recurrent infections of the respiratory tract in humans. Previous indirect evidence suggested that a strain-specific immune response occurs following infection and that this immune response is directed at an immunodominant epitope on the bacterial surface. To test this hypothesis, mice and rabbits were immunized with whole cells of a strain of NTHI and the antiserum was characterized to identify the antigens to which antibodies were directed. All animals made a prominent antibody response to the loop 5 region of the P2 molecule, which is the major outer membrane protein. Rabbit serum showed complement-dependent bactericidal activity. Adsorption of the immune serum with the loop 5 fusion peptide removed bactericidal activity and also abolished reactivity to P2 detected by an immunoblot assay, an enzyme-linked immunosorbent assay, and a radioimmunoprecipitation assay. These data indicate that immunization with whole cells of NTHI results in a prominent antibody response which is directed at epitopes on the loop 5 region of the P2 molecule. Thus, a strain-specific immune response to NTHI occurs as a result of the expression of an immunodominant epitope on the P2 molecule.

Animals↗