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Biomedical subjects

E J Hansen

Publications and source records attributed to E J Hansen.

At least 127 records · Page 7Linked to original sources
PubMed1987

Conservation of epitopes in the oligosaccharide portion of the lipooligosaccharide of Haemophilus influenzae type b.

The antigenic characteristics of the lipooligosaccharide (LOS) of Haemophilus influenzae type b (Hib) were examined in strains obtained over an extended period of time. These Hib strains were isolated from patients with systemic Hib disease in Dallas, Tex., over a 20-year period and in New York City between 1941 and 1956. The antigenic characteristics of the LOS of these Hib strains were examined by using a set of four murine monoclonal antibodies directed against epitopes present in the oligosaccharide portion of the LOS molecule. The same basic set of LOS antigenic determinants that is expressed by recent Hib isolates was also found to be present in this collection of Hib strains spanning a 40-year period. Some variation with time was detected in the distribution of the systemic disease isolates among four Hib LOS antigenic groups; however, only 2 of 188 Hib isolates failed to react with a set of two LOS-specific monoclonal antibodies. Therefore, little variation has occurred among Hib strains with regard to the LOS epitopes defined by these monoclonal antibodies over a considerable period of time.

Antibodies, Bacterial↗
PubMed1987

Dissociation of virulence and protection from infection by mutant analysis in Haemophilus influenzae type b.

A Haemophilus influenzae type b (Hib) outer membrane protein with an apparent molecular weight of 98,000 (98K) previously has been shown to react with antibodies that protect against experimental Hib disease. A mutant lacking the ability to synthesize this 98K protein was produced by chemical mutagenesis and identified in a colony blot-radioimmunoassay by its failure to react with a 98K protein-specific monoclonal antibody. DNA from this mutant was used to produce a 98K protein-negative transformant of the wild-type parental strain. Comparison of the relative degree of virulence of the parental strain and the 98K protein-negative transformant in an animal model system revealed no differences in the abilities of these two strains to produce bacteremia after intranasal challenge. These results indicate that the Hib surface-exposed 98K outer membrane protein that reacts with protective antibodies plays no detectable role in the expression of virulence by Hib in an animal model system.

Animals↗
PubMed1987

Haemophilus influenzae type b lipooligosaccharide: stability of expression and association with virulence.

Spontaneous antigenic and phenotypic variations in the lipooligosaccharide (LOS) of two strains of Haemophilus influenzae type b (Hib) were previously shown to be associated with changes in virulence (A. Kimura and E.J. Hansen, Infect. Immun. 51:69-79, 1986). The goal of the present study was to define further the stability of LOS expression by this pathogen and the role of Hib LOS in virulence. Variation in LOS antigenic reactivity, as detected with LOS-specific monoclonal antibodies, was observed in 3 of 30 Hib strains after single-colony passage. When large numbers of individual colonies from seven other Hib strains were screened, however, spontaneous LOS antigenic variation was detected in all of the strains. Antigenic variation was not consistently associated with an altered LOS phenotype, as determined by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and silver staining of LOS preparations. Changes in the LOS antigenic phenotype were correlated with altered virulence potential in two strains. In these strains, acquisition of reactivity with certain LOS-directed monoclonal antibodies was associated with the synthesis of a higher-molecular-weight LOS, enhanced virulence, and increased resistance to serum killing involving the classical complement pathway.

Animals↗
PubMed1986

Antigenic and phenotypic variations of Haemophilus influenzae type b lipopolysaccharide and their relationship to virulence.

Haemophilus influenzae type b (Hib) strains NO100 and COL10 were found to produce bacteremia in infant rats at a much lower frequency than other Hib strains previously tested. These relatively avirulent strains were the only Hib strains among 200 clinical isolates examined to date which failed to react with two Hib lipopolysaccharide (LPS)-specific monoclonal antibodies (MAbs). LPS analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that strains NO100 and COL10 possessed LPS which migrated faster than the LPS of Hib strains that reacted with one of the two or with both of these MAbs. These observations suggested that the relative lack of virulence of strains NO100 and COL10 might be related to their unusual LPS phenotype. To determine whether alteration of LPS structure would affect the virulence of these strains, we identified and isolated isogenic LPS antigenic variants of strains NO100 and COL10 using the LPS-specific MAbs 4C4 and 5G8 in a colony blot radioimmunoassay. Antigenic variation of LPS was found to occur spontaneously in these two strains at a relatively high frequency in terms of both acquisition and loss of MAb reactivity (ca. 0.2 to 16.7%). LPS antigenic variants of strains NO100 and COL10 reactive with both MAbs 4C4 and 5G8 (4C4+ 5G8+) were more virulent in the infant rat model than their respective 4C4- 5G8- parental strains (P less than 0.01). An antigenic variant of COL10 reactive with only MAb 4C4 (4C4+ 5G8-) was also significantly more virulent than its 4C4- 5G8- parent. These LPS antigenic variants with increased virulence synthesized altered LPS molecules which possessed apparent molecular weights higher than those of the LPS of the parental strains. Increased resistance of strain NO100 to the bactericidal activity of normal infant rat serum was associated with changes in LPS structure, while strain COL10 and its LPS variants were all uniformly resistant to serum bactericidal activity. Our results demonstrate that (i) spontaneous antigenic and phenotypic variation of LPS occurs at a relatively high frequency in some strains of Hib; (ii) the higher-molecular-weight type of LPS is associated with the full expression of Hib virulence; (iii) LPS phenotype may not correlate with Hib serum resistance; and (iv) serum resistance of Hib is not an accurate indicator of virulence.

Animals↗
PubMed1985

A minor high-molecular-weight outer membrane protein of Haemophilus influenzae type b is a protective antigen.

Cell surface-exposed antigenic determinants of several high-molecular-weight outer membrane proteins of Haemophilus influenzae type b (Hib) have been shown to be consistently immunogenic in human infants convalescing from Hib meningitis. A monoclonal antibody (mab), 6G12, directed against one of these cell surface-exposed outer membrane proteins that has an apparent molecular weight of 98,000 (98K) was identified by radioimmunoprecipitation analysis. Of 120 clinical isolates of Hib, 83 were found to possess antigenic determinants which reacted with mab 6G12 in a colony blot-radioimmunoassay procedure, indicating that the antigenic determinant recognized by mab 6G12 is present in the majority of Hib strains. A different radioimmunoassay, which uses whole Hib cells as antigen, confirmed that strains reactive with mab 6G12 in the colony blot-radioimmunoassay procedure possessed a cell surface-exposed and antibody-accessible antigenic determinant recognized by this mab. Hib strains which did not react with mab 6G12 were found to lack a 98K protein. Passive immunization with mab 6G12 reduced the level of bacteremia that developed in infant rats challenged with the homologous Hib strain against which this mab was raised. In contrast, no protection was observed when the challenge strain was one which lacks the antigenic determinant recognized by mab 6G12. Radioimmunoprecipitation analysis of sera from human infants convalescing from Hib meningitis detected an antibody response directed against the 98K protein. The protection against experimental Hib disease provided by antibody to the 98K protein, the immunogenicity of this protein in human infants, and its presence in a majority of Hib strains indicate that the 98K outer membrane protein may have potential for vaccine development.

Animals↗
PubMed1985

Effect of systemic immunization on pulmonary clearance of Haemophilus influenzae type b.

The effect of systemic immunization on pulmonary clearance of Haemophilus influenzae type b (Hib) was studied in a mouse model system. Immunization of mice by intraperitoneal injection of viable Hib cells resulted in the appearance of Hib-directed antibodies in both serum and bronchoalveolar lavage fluid. The development of this Hib-directed antibody activity was associated with significant enhancement of early pulmonary clearance of Hib. Systemic immunization did not affect the recruitment of polymorphonuclear leukocytes to the alveoli, suggesting that the enhanced clearance of Hib observed in immunized animals was due to specific antibodies which promote either phagocytosis or extracellular killing of Hib. The spectrum of Hib-directed antibody specificities detected in sera from immunized animals was essentially identical to that detected in bronchoalveolar lavage fluids from these same animals. Similarly, intravenous administration of an immunoglobulin G monoclonal antibody specific for Hib lipopolysaccharide resulted in the subsequent appearance of this antibody in the alveolar spaces where it enhanced pulmonary clearance of Hib. This study shows that this mouse model system can be used to measure the effect of both active and passive immunization on the clearance of Hib from the lower respiratory tract.

Animals↗
PubMed1985

Coprecipitation of lipopolysaccharide and the 39,000-molecular-weight major outer membrane protein of Haemophilus influenzae type b by lipopolysaccharide-directed monoclonal antibody.

The major outer membrane protein of Haemophilus influenzae type b (Hib) with an apparent molecular weight of 39,000 (39K) was purified from three different Hib strains and was shown to be free from detectable contamination with other proteins. However, these purified 39K protein preparations were found to contain Hib lipopolysaccharide (LPS). Immunization of rats with these 39K protein preparations resulted in the production of antisera containing both 39K protein-directed and LPS-directed antibodies, as determined by Western blot analysis. The reactivity pattern of the LPS-directed serum antibodies with different Hib strains was identical to the reactivity of these Hib strains with a set of monoclonal antibodies (mabs) previously shown to immunoprecipitate the 39K protein in a radioimmunoprecipitation (RIP) system. Examination of the antigenic specificities of the 39K protein-immunoprecipitating mabs by using Western blot analysis showed that these mabs were actually directed against Hib LPS. RIP analysis of 125I-labeled Hib cells and 32P-labeled Hib cells revealed that the 39K protein and LPS existed as a complex in a RIP system, which resulted in the coprecipitation of both antigens by LPS-directed mabs. The interaction between LPS and the 39K protein was highly selective for this protein and did not involve other outer membrane proteins. The LPS/39K protein complex could be reconstituted by mixing purified LPS and purified 39K protein; it could also be reconstituted with 39K protein from one Hib strain and LPS from another Hib strain. These findings have necessitated the reinterpretation of previous studies involving the 39K protein-immunoprecipitating mabs. Of primary importance is the fact that the demonstrated immunoprotective ability of a 39K protein-immunoprecipitating mab (E. J. Hansen, S. M. Robertson, P. A. Gulig, C. F. Frisch, and E. J. Haanes, Lancet i:366-368, 1982) must now be regarded as evidence that antibody directed against Hib LPS can be protective against experimental Hib disease.

Animals↗
PubMed1985

Role of C5 and recruited neutrophils in early clearance of nontypable Haemophilus influenzae from murine lungs.

We used a mouse model system to investigate the pulmonary defense mechanisms involved in clearance of nontypable Haemophilus influenzae from the lower respiratory tract. The importance of the C5 complement protein molecule in polymorphonuclear leukocyte (PMN) recruitment was studied by using congenic C5-sufficient B10.D2/nSn (C5+) and C5-deficient B10.D2/oSn (C5-) mice. The C5+ and C5- mice were inoculated with saline or nontypable H. influenzae via an endobronchial catheter. Clearance of bacteria was studied by using quantitative lung cultures. Bronchoalveolar lavage was performed at several time intervals. The number of cells in the lavage fluid were counted, and chemotactic activity was assayed in lavage fluid by the leading front technique, using human PMN in modified Boyden chambers. Pulmonary clearance of bacteria was significantly impaired in the absence of C5 (P less than 0.05). The C5+ mice recruited significantly more PMN after challenge with nontypable H. influenzae than C5- mice did (P less than 0.05), but significant PMN recruitment occurred in C5- mice. Similarly, although chemotactic activity was present in both C5+ and C5- mice, significantly more intraalveolar chemotactic activity was noted in C5+ mice than in C5- mice (P less than 0.05). The C5 molecule yields important chemotaxins during this early time period, but other chemotaxins are also present within the alveoli, demonstrating the redundancy of the inflammatory response after pulmonary challenge with nontypable H. influenzae. Nitrogen mustard-induced neutropenic animals were studied to evaluate the functional importance of PMN in pulmonary clearance of nontypable H. influenzae. Pulmonary clearance of nontypable H. influenzae was significantly impaired in neutropenic animals (P less than 0.05). Our results indicate that the prompt appearance of PMN in lungs is crucial for early clearance of nontypable H. influenzae.

Animals↗
PubMed1985

Cloning and surface expression in Escherichia coli of a structural gene encoding a surface protein of Haemophilus influenzae type b.

Recombinant DNA technology was used to clone a gene coding for a surface protein of Haemophilus influenzae type b (Hib) into Escherichia coli. Chromosomal DNA from a clinical isolate of Hib was cleaved with EcoRI and ligated into plasmid vectors containing three different translational reading frames. E. coli carrying recombinant plasmids were screened in a colony blot-radioimmunoassay system by using murine monoclonal antibodies (mabs) directed against cell surface-exposed proteins of Hib. mab 7B2, which is specific for a Hib surface protein with an apparent molecular weight of 27,000 (27K), reacted with several recombinant strains of E. coli. Restriction analysis revealed the presence of a 9.1-kilobase DNA insert in each of these recombinant plasmids and also determined that both transcription and translation of the Hib gene(s) coding for the 7B2-reactive antigen were not dependent on the lac operator and promoter of the vectors. Radioimmunoprecipitation and Western blot analyses showed that the antigenic determinant recognized by mab 7B2 in these recombinant E. coli was present in a 27K protein. In addition, this 27K protein was shown to be both localized on the surface of these E. coli cells and accessible to antibody.

Antigens, Surface↗
PubMed1984

Monoclonal antibodies reactive with all strains of Haemophilus ducreyi.

We have isolated plasma cell hybridomas which secrete monoclonal antibodies directed against Haemophilus ducreyi. Two of these monoclonal antibodies recognize all strains of H. ducreyi tested to date and are capable of detecting the presence of H. ducreyi in skin lesions produced by this pathogen in experimental animals. These monoclonal antibodies which react with apparently all strains of H. ducreyi have the potential to be developed into a rapid immunodiagnostic test for chancroid.

Animals↗
PubMed1984

Immunogenic proteins in cell-free culture supernatants of Haemophilus influenzae type b.

Cell-free culture supernatant (CFCS) prepared from Haemophilus influenzae type b (Hib) was examined for the presence of soluble Hib proteins. Two proteins with apparent molecular weights of 100,000 (100K) and 116K were predominant in the CFCS, and antibodies directed against these proteins could be detected by radioimmunoprecipitation or Western blot analyses of serum from adult rats immunized with Hib. Radioimmunoprecipitation analyses and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these two proteins demonstrated that the 100K CFCS protein was also present on the cell surface of Hib, whereas the 116K CFCS protein was only detectable in culture supernatants. Both the 100K and 116K CFCS proteins were immunogenic in human infants with Hib meningitis and in infant rats systemically infected with Hib. In addition, the first detectable antibodies produced in these Hib-infected rats against Hib proteins were specific for the 100K protein in both its CFCS and cell-associated forms. These two CFCS proteins were also immunogenic in rats immunized with CFCS in the absence of Hib infection. Monoclonal antibody directed against the 100K protein reacted with 34 of 55 Hib strains examined by using a colony blot radioimmunoassay. The immunogenicity of the 100K and 116K CFCS proteins suggests that one or both of these proteins may have potential for vaccine development, either by themselves or covalently coupled to Hib capsular polysaccharide.

Animals↗
PubMed1984

Pulmonary clearance of encapsulated and unencapsulated Haemophilus influenzae strains.

A mouse model system was employed to investigate the temporal pattern of pulmonary clearance of Haemophilus influenzae and to evaluate the effect of the type b polysaccharide capsule on this clearance pattern. The lungs of BALB/c mice were inoculated with boluses of several different H. influenzae strains via an endobronchial catheter. A fully encapsulated H. influenzae type b strain multiplied readily in the lungs for at least 6 h and then was eventually cleared from the lungs over the next 18 h. The pulmonary clearance pattern obtained with an unencapsulated variant of this H. influenzae type b strain was identical to that obtained with the fully encapsulated parent strain. Two nontypable H. influenzae strains isolated by transtracheal aspiration of patients with acute H. influenzae pneumonia also multiplied in the lung and resisted significant clearance for at least 6 h after inoculation. Bolus deposition of either H. influenzae type b or nontypable H. influenzae in the lungs resulted in an eventual influx of polymorphonuclear leukocytes into the alveoli. The observed delay in clearance of all these strains suggests that resident host defense mechanisms must be augmented for clearance to occur. Furthermore, these data indicate that one or more factors other than the Haemophilus capsule are important bacterial determinants of pulmonary clearance of H. influenzae.

Animals↗
PubMed1983

Hemadsorption and virulence are separable properties of Mycoplasma pneumoniae.

A selective enrichment technique was used to isolate a hemadsorption-positive revertant of a hemadsorption-negative mutant strain of Mycoplasma pneumoniae. This hemadsorption-positive revertant was shown to have simultaneously regained both the ability to attach to neuraminidase-sensitive receptors on the tracheal ring respiratory epithelium in vitro and the ability to synthesize three virulent-strain-specific proteins which were not synthesized by the hemadsorption-negative mutant. Despite the persistence of the revertant in hamster lung tissue for 9 to 12 weeks postinfection, no cytopathology was observed. Intranasal inoculation of the revertant provided limited protection against a challenge dose of virulent M. pneumoniae.

Adhesiveness↗
PubMed1983

A set of two monoclonal antibodies specific for the cell surface-exposed 39K major outer membrane protein of Haemophilus influenzae type b defines all strains of this pathogen.

Six murine plasma cell hybridomas producing monoclonal antibodies (mabs) directed against the 39,000-molecular-weight (39K) major outer membrane protein of Haemophilus influenzae type b were employed in the antigenic analysis of the 39K protein. The initial characterization of the mabs by radioimmunoprecipitation analysis showed that four of these mabs reacted with antigenic determinants of the 39K protein that are exposed on the bacterial cell surface and accessible to antibody. The other two mabs reacted with antigenic determinants of the 39K protein that are either not exposed on the H. influenzae type b cell surface or not accessible to antibody (internal determinants). A total of 126 clinical isolates of H. influenzae type b obtained from pediatric research centers throughout the United States were examined for reactivity with the six mabs by using a solid-phase radioimmunoassay in which bacterial colony growth from agar plates was placed on filter paper and used as antigen. The reactivities of these strains with two of the mabs recognizing cell surface-exposed antigenic determinants of the 39K protein were used to divide the 126 strains into four different groups. Group 1 strains reacted with mab 12D9, group 2 strains reacted with mab 4C4, group 3 strains reacted with both mabs 12D9 and 4C4, and group 4 strains (only one was found) did not react with either mab. The reactivities of two other mabs recognizing cell surface-exposed antigenic determinants of the 39K protein were used to further divide the four groups into eight subgroups. A single mab recognizing an internal antigenic determinant of the 39K protein reacted with every H. influenzae type b strain examined in this study. These data indicate that only limited antigenic heterogeneity exists among the cell surface-exposed antigenic determinants of the 39K outer membrane proteins among H. influenzae type b strains and that at least one internal antigenic determinant of the 39K protein is universally present in all H. influenzae type b strains. Radioimmunoprecipitation analysis also demonstrated that H. influenzae type b strains which lacked a 39K major outer membrane protein possessed a 38K major outer membrane protein which reacted with the anti-39K mabs, indicating that the 38K and 39K outer membrane proteins of different H. influenzae type b strains are antigenically related.

Animals↗
PubMed1982

Immunoprotection of rats against Haemophilus influenzae type B disease mediated by monoclonal antibody against a haemophilus outer-membrane protein.

Infant rats passively immunised with a murine monoclonal antibody against a cell-surface-exposed outer-membrane protein of Haemophilus influenzae by b (Hib) were protected against systemic Hib disease induced by intraperitoneal injection of virulent Hib cells. The immunoprotection mediated by this monoclonal antibody was specific for Hib strains carrying the protein antigenic determinant that it recognised; the antibody gave no protection against disease when passively immunised animals were challenged with a heterologous Hib strain. The immunoprotection given by an antibody against a Hib outer-membrane protein suggests that Hib outer-membrane proteins have potential for development as Hib vaccines.

Animals↗
PubMed1982

Monoclonal antibodies directed against a cell surface-exposed outer membrane protein of Haemophilus influenzae type b.

Monoclonal antibodies directed against several different Haemophilus influenzae type b outer membrane proteins with apparent molecular weights of 45,000, 39,000, and 37,000 were identified by a radioimmunoprecipitation method. Five monoclonal antibodies, including both immunoglobulin G and M isotypes, were specific for the same H. influenzae type b major outer membrane protein (39,000 molecular weight). One of these immunoglobulin G monoclonal antibodies (6A2) was shown to be directed against a cell surface-exposed antigenic determinant of the 39,000-molecular-weight protein, whereas the other monoclonal antibodies directed against this same protein were apparently specific for antigenic determinants not exposed on the H. Influenzae type b cell surface. The cell surface-exposed protein antigenic determinant recognized by monoclonal antibody 6A2 was not unique to the H. influenzae type b strain used as the source of outer membrane vesicles for generating immune spleen cells, but was found in a majority of independently isolated strains of H. influenzae type b. These data indicate that there is antigenic cross-reactivity among H. influenzae type b strains with regard to cell surface-exposed proteins.

Antibodies, Monoclonal↗