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T F Murphy

Publications and source records attributed to T F Murphy.

At least 55 records · Page 3Linked to original sources
PubMed1997

Specific binding of Haemophilus influenzae to minor gangliosides of human respiratory epithelial cells.

Gangliosides are sialylated glycosphingolipids that serve as receptors for various bacteria. To investigate endogenous gangliosides of human respiratory epithelial cells as potential receptors for Haemophilus influenzae, three strains, including nontypeable H. influenzae (NTHI) 1479, and isogenic fimbriated (f+) and nonfimbriated (f0) H. influenzae type b 770235, were 3H labeled and overlaid on two-dimensional thin-layer chromatography (TLC) plates containing either purified HEp-2 gangliosides or murine brain gangliosides. NTHI 1479 bound exclusively to two distinct minor ganglioside doublets, with mobilities near that of GM1. These minor gangliosides comprised only 14.2 and 9.4% of the total, respectively. NTHI 1479 also bound to a distinct ganglioside of human macrophages whose chromatographic mobilities closely resemble those of one of the NTHI-binding gangliosides of HEp-2 cells. H. influenzae type b 770235 f+ and f0 each bound to a different minor HEp-2 ganglioside doublet, with proportionately weaker affinity for a major ganglioside doublet. Remarkably, none of the three strains bound to any murine brain gangliosides. Moreover, when 80 to 90% of sialic acid residues were enzymatically removed from HEp-2 gangliosides, NTHI 1479 binding was proportionately impaired, compared with untreated controls. Our findings support a role for specific gangliosides of specific cells as receptors for H. influenzae strains. Our findings further demonstrate that individual minor gangliosides possess unique biological properties.

Bacterial Adhesion↗
PubMed1997

Antigenic characterization and analysis of the human immune response to outer membrane protein E of Branhamella catarrhalis.

Outer membrane protein E (OMP E) is a 50-kDa major OMP of Branhamella catarrhalis. Polyclonal antisera and four monoclonal antibodies (MAbs) to OMP E were generated to study its antigenic structure. All antibodies recognized epitopes in all 19 B. catarrhalis strains tested by immunoblot assays. By flow cytometry, it was determined that MAbs 1B3 and 9G10d recognized epitopes which are expressed on the surface of the intact bacterium, while MAbs IC11 and 7C10 recognized epitopes which were buried within the outer membrane. A competitive enzyme-linked immunosorbent assay showed that MAbs 1B3 and 9G10d recognize the same or closely related epitopes. Proteinase K treatment of whole bacterial cells revealed that MAbs 1B3 and 9G10d recognize a surface-exposed epitope located in the 17-kDa region towards the amino terminus of OMP E. The human serum and mucosal antibody responses to OMP E in adults with chronic bronchitis were studied. A majority of these patients had immunoglobulin A to OMP E in sputum supernatants. None of ten adults who experienced lower respiratory tract infections due to B. catarrhalis demonstrated a clear-cut rise in antibody titer to OMP E in serum or sputum supernatant. This study has demonstrated that OMP E has at least one surface-exposed epitope which is highly conserved among strains of B. catarrhalis and which is located in the amino-terminal 184 amino acids of the molecule.

Adult↗
PubMed1997

Antigenic heterogeneity and molecular analysis of CopB of Moraxella (Branhamella) catarrhalis.

Outer membrane protein (OMP) CopB, an iron-repressible 81-kDa major OMP of Moraxella (Branhamella) catarrhalis has been a major focus of investigation. To assess CopB as a potential vaccine antigen, we elucidated the degree of antigenic and sequence heterogeneity in this protein among strains of M. catarrhalis. Two monoclonal antibodies, 1F5 and 2.9F, which bind to surface-exposed epitopes on CopB recognized 60 and 70% of the strains, respectively. The degree of sequence heterogeneity in CopB was assessed by cloning and sequencing the CopB gene from two different strains of M. catarrhalis and comparing with the published sequence. There was 92 to 96% homology between the sequences at the nucleotide level and 90 to 95% homology at the amino acid level. The variability in the protein sequence is confined mainly to three moderately variable regions. Restriction fragment length polymorphism (RFLP) analysis of the CopB genes obtained from 20 diverse strains by PCR was performed. Ninety percent of the potential restriction sites in the constant regions and 47% of the potential restriction sites in the variable regions were present in the 20 strains, indicating that the pattern of variable and constant areas in the CopB gene is a general pattern among strains of M. catarrhalis. We conclude that the CopB gene is largely conserved among strains of M. catarrhalis and contains discrete regions which show moderate heterogeneity among strains.

Amino Acid Sequence↗
PubMed1996

Isolation and characterization of the Haemophilus influenzae tolQ, tolR, tolA and tolB genes.

The tolQ, R, A and B genes have been isolated from the DNA of Haemophilus influenzae and sequenced. The deduced amino acid (aa) sequence of the H. influenzae TolQ, TolR, TolA and TolB show 67, 63, 41 and 62% identity with Escherichia coli TolQRAB proteins, respectively. These four proteins are involved in transport of colicins and phages across the cell envelope. The translational stop codon of TolB (the last gene in the cluster) is 23 bases upstream of the start codon of the P6 lipoprotein gene. Primer extension and Northern blot analysis revealed that the start of the P6 transcript is within the tolB gene. Nucleotide sequence (nt) analysis of the entire tolQRABP6 region shows a transcriptional terminator immediately downstream of the P6 gene. The tolQRABP6 gene cluster of H. influenzae may thus constitute an operon.

Amino Acid Sequence↗
PubMed1996

A monoclonal antibody to human macrophage gangliosides inhibits macrophage migration.

Gangliosides have diverse immunoregulatory properties. The gangliosides endogenous to macrophages may have immunoregulatory properties that distinguish them from other gangliosides. Gangliosides have been indirectly implicated in macrophage migration as putative cell surface receptors for migration inhibitory factor (MIF). In this study, a monoclonal antibody to human macrophage gangliosides (antibody 25F4) was developed and characterized. This is the first report of the development of monoclonal antibodies to gangliosides of macrophages of any species. Thin-layer chromatographic immunostaining indicated that antibody 25F4 recognized major gangliosides of human macrophages but did not recognized those previously identified as containing fucose. Immunofluorescent surface labeling of viable human macrophages indicated that antibody 25F4 recognized a surface-accessible epitope, present on all cells, and that this was abolished with lipid depletion of macrophage membranes. This epitope was not present on several human nonmacrophage cells. Finally, human macrophages pretreated with antibody 25F4 demonstrated striking inhibition of migration of an agarose droplet assay, whereas an irrelevant monoclonal antibody or monoclonal antibodies to nonganglioside surface epitopes of human macrophages had no effect on migration. Migration inhibition occurred even though antibody 25F4 was removed from the extracellular milieu and was not due to formation of cellular aggregates. These studies support a role for human macrophage gangliosides in macrophage migration.

Antibodies, Monoclonal↗
PubMed1996

Binding between outer membrane proteins of nontypeable Haemophilus influenzae and human nasopharyngeal mucin.

Bacterial colonization of the epithelial cells precedes infection. Mucins of the epithelial cell secretions modulate bacterial colonization. This study was designed to understand the mechanism of mucin-bacterial interactions and in particular binding between nontypeable Haemophilus influenzae and nasopharyngeal mucin(s). In an overlay assay, binding appears to be mediated by outer membrane proteins P2 and P5 of bacteria and sialic acid-containing oligosaccharides of mucin.

Bacterial Outer Membrane Proteins↗
PubMed1996

Branhamella catarrhalis: epidemiology, surface antigenic structure, and immune response.

Over the past decade, Branhamella catarrhalis has emerged as an important human pathogen. The bacterium is a common cause of otitis media in children and of lower respiratory tract infections in adults with chronic obstructive pulmonary disease. B. catarrhalis is exclusively a human pathogen. It colonizes the respiratory tract of a small proportion of adults and a larger proportion of children. Studies involving restriction enzyme analysis of genomic DNA show that colonization is a dynamic process, with the human host eliminating and acquiring new strains frequently. The surface of B. catarrhalis contains outer membrane proteins, lipooligosaccharide, and pili. The genes which encode several outer membrane proteins have been cloned, and some of these proteins are being studied as potential vaccine antigens. Analysis of the immune response has been limited by the lack of an adequate animal model of B. catarrhalis infection. New information regarding outer membrane structure should guide studies of the human immune response to B. catarrhalis. Immunoassays which specifically detect antibodies to determinants exposed on the bacterial surface will elucidate the most relevant immune response. The recognition of B. catarrhalis as an important human pathogen has stimulated research on the epidemiology and surface structures of the bacterium. Future studies to understand the mechanisms of infection and to elucidate the human immune response to infection hold promise of developing new methods to treat and prevent infections caused by B. catarrhalis.

Adult↗
PubMed1995

Outer membrane protein CD of Branhamella catarrhalis: sequence conservation in strains recovered from the human respiratory tract.

Branhamella catarrhalis causes lower respiratory tract infections in patients with chronic obstructive pulmonary disease. The outer membrane protein CD (OMP-CD) of B. catarrhalis is a major, heat-modifiable OMP. The goals of this study are to characterize the degree of conservation of OMP-CD among strains and to investigate if OMP-CD maintains its homogeneity under the effect of host immune selective pressure. Isolates of B. catarrhalis were collected prospectively from patients with bronchiectasis and chronic bronchitis. We studied the OMP-CD gene by analysis of PCR restriction fragment length polymorphisms (PCR-RFLP) and further determined DNA sequence of the CD gene of eight selected isolates. Five patterns of PCR-RFLP of the OMP-CD gene were observed among all isolates when the gene was digested with Sau3AI. The sequence analysis revealed a high degree of homogeneity in OMP-CD among strains of B. catarrhalis. Three regions of OMP-CD with minimal sequence heterogeneity were identified. The sequences of the OMP-CD gene of isolates collected from patients colonized with the same strain for up to 6 months was identical. These observations establish that the OMP-CD of B. catarrhalis recovered from clinical isolates is highly conserved among strains.

Bacterial Outer Membrane Proteins↗
PubMed1995

A comparison of survival at different degrees of hemorrhagic shock in germ-free and germ-bearing rats.

We have previously reported superior survival after one level of hemorrhagic shock in germ-free (GF) rats compared with germ-bearing (GB) rats. The objective of this study was to determine the effect of the GF state on survival at different degrees of hemorrhagic shock. GF and GB rats were bled to a mean arterial blood pressure of 30 mmHg. Shock was terminated after 10, 20, 40, or 80% of the maximum shed blood volume was reabsorbed spontaneously. Both shock time and time to decompensation were significantly longer in GF rats (p < .05). Comparative survival was greater for GF rats at most levels of shock (p < .01). This superiority in survival was greatest at moderate shock levels and decreased at severe shock levels. There may be several reasons for the increased tolerance of GF animals to hemorrhagic shock such as metabolic or immunologic variations. It is hard to avoid the fact, however, that the most notable difference between the GF and GB rat is the presence or absence of bacteria.

Animals↗
PubMed1995

Serum antibodies to outer membrane proteins (OMPs) of Moraxella (Branhamella) catarrhalis in patients with bronchiectasis: identification of OMP B1 as an important antigen.

Moraxella (Branhamella) catarrhalis is a common cause of lower respiratory tract infections in adults and of otitis media in children. Little is known about the human immune response to this bacterium. In this study, immunoblot assays were performed to detect serum immunoglobulin G antibodies directed at purified outer membrane of M. catarrhalis. Twelve serum samples, two each from six patients with bronchiectasis who were persistently colonized with this organism, were tested with their homologous M. catarrhalis sputum isolates. In all the sera, the most prominent and consistent antibody response was to a minor 84-kDa outer membrane protein, OMP B1. Immunoblot adsorption assays show that these antibodies recognize surface exposed epitopes on OMP B1. Further analysis of human serum antibodies eluted from the surface of intact bacterial cells shows that these surface-exposed epitopes on OMP B1 are heterogeneous among strains of M. catarrhalis. OMP B1 is therefore an important OMP antigen on the surface of M. catarrhalis for the human immune response to infection by this bacterium.

Adult↗
PubMed1995

Dynamics of respiratory tract colonization by Branhamella catarrhalis in bronchiectasis.

Branhamella catarrhalis is increasingly recognized as a lower respiratory tract pathogen, particularly in chronic lung diseases. This project defines a population of patients in whom the dynamics of colonization and infection caused by this organism could be studied. A method employing pulsed field gel electrophoresis (PFGE) of genomic DNA was developed. Twenty-eight patients with bronchiectasis followed prospectively for 26.8 mo (mean) were seen monthly or bimonthly and at the time of a purulent exacerbation. Quantitative bacterial cultures were performed on sputum obtained at each visit. Six of 28 had B. catarrhalis isolated repeatedly. Viable numbers of B. catarrhalis were similar to other bacterial pathogens. Restriction fragment length polymorphism (RFLP) analysis of chromosomal DNA using PFGE was performed on 37 of the 47 isolates recovered. Each patient was colonized by two to four strains with different RFLP patterns. Duration of colonization by the same strain was 2.3 mo (mean). Strain acquisition did not correlate with exacerbation, antibiotic therapy, or season. We conclude that (1) a subset of bronchiectatic patients is colonized with B. catarrhalis, (2) RFLP is a sensitive tool to study strain acquisition, and (3) acquisition and clearance of B. catarrhalis from the respiratory tract is a dynamic process.

Adult↗
PubMed1994

Mapping of a strain-specific bactericidal epitope to the surface-exposed loop 5 on the P2 porin protein of non-typeable Haemophilus influenzae.

The P2 protein is the major outer-membrane protein of non-typeable Haemophilus influenzae (NTHI) and shows extreme heterogeneity among strains. Based on the analysis of antigenic structure, the P2 protein consists of eight potentially surface-exposed loops. Previous studies of monoclonal antibodies (mABs) to a single strain of NTHI showed that P2 contains potentially immunodominant epitopes in loop 5 of the molecule. The goal of the current work is to test the hypothesis that strain-specific and potentially immunodominant epitopes are located in loop 5 of P2 in other strains of NTHI as well. Gene fragments which encode peptides of loop 5 of strains 2019 and 5657 were cloned into an expression vector and subjected to immunoassays with mABs which recognize surface-exposed, bactericidal, strain-specific epitopes. Each mAB recognized loop 5 of the P2 protein of the homologous strain. Analysis of mutant clones with minor amino acid changes showed a loss of reactivity with the mABs. These observations indicate that loop 5 of the P2 molecule contains strain-specific, abundantly expressed surface-exposed epitopes. This further supports the hypothesis that loop 5 is an immunodominant region of the P2 molecule.

Adult↗
PubMed1994

Health care workers with HIV and a patient's right to know.

Accidental human immunodeficiency virus (HIV) infection of patients in health care settings raises the question about whether patients have a right to expect disclosure of HIV/AIDS diagnoses by their health workers. Although such a right-and the correlative duty to disclose-might appear justified by reason of standards of informed consent, I argue that such standards should only apply to questions of risks of and barriers to HIV infection involved in a particular medical treatment, not to disclosure of personal diagnoses. Because the degree of risk of HIV infection is low and disclosure would also have damaging consequences for health workers, and because patient protection is available in other ways, it is argued that no such generalized right should be recognized.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Purification and characterization of a high-molecular-weight outer membrane protein of Moraxella (Branhamella) catarrhalis.

Moraxella (Branhamella) catarrhalis is an important bacterial cause of otitis media in children and lower respiratory tract infections in adults. In this study, we describe the presence of a novel high-molecular-weight outer membrane protein (HMW-OMP). This protein varies from 350 to 720 kDa in apparent molecular mass among strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein was detected on SDS-PAGE in 13 of 14 strains tested. We developed a monoclonal antibody and polyclonal antisera to this protein. In immunoblot assays, the protein was present in all 14 strains tested. The immunoblot assays suggest that the protein has at least one epitope that is conserved among strains. A purification method using anion-exchange chromatography is described. Treatment of outer membrane preparations and purified protein by heat and reducing agents did not change the apparent molecular mass of the HMW-OMP. Formic acid treatment of outer membrane preparations and purified HMW-OMP produced a single band with an apparent molecular mass of 120 to 140 kDa. We postulate that this may be the monomer of an oligomeric protein. The HMW-OMP, which varies in molecular mass among strains and is antigenically conserved, will be studied further to determine its role in the human immune response and may be useful as a marker in studying strain acquisition in patients.

Animals↗