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E J Hansen

Publications and source records attributed to E J Hansen.

At least 73 records · Page 4Linked to original sources
PubMed1993

Outer membrane protein binding sites of complement component 3 during opsonization of Haemophilus influenzae.

Complement component 3 (C3) binding to Haemophilus influenzae type b (Hib) is an important step in host defense against invasive disease, but the details of this process remain poorly understood. We have shown that the P1 and P2 outer membrane proteins (OMPs) serve as binding sites for C3 on serum-opsonized Hib. Whole-cell lysates of opsonized Hib were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were transferred to nitrocellulose. Immunoblot analysis with monoclonal antibodies (MAbs) to the 49-kDa P1 and 39-kDa P2 OMPs demonstrated high-molecular-weight bands that were not present when the bacteria were opsonized with heat-inactivated or methylamine-treated serum. Immunoblot analysis with MAbs to the 98- or 16-kDa (P6) OMPs did not reveal additional bands. An unencapsulated Hib mutant still lacked C3 bound to the 98-kDa or P6 OMP, indicating that the absence of C3 binding to these proteins was not the result of epitope masking by the capsule. Studies with MAbs to C3 fragments confirmed that the anti-P1- and anti-P2-reactive bands were C3 fragments bound to these OMPs. The molecular weights of proteins reactive to anti-OMP and anti-C3 antibodies indicated that multiple C3 fragments may be bound to P1 or that C3 may be bound to P2 multimers. Finally, the presence of other anti-C3-reactive proteins indicated that several other proteins serve as C3 targets during the opsonization of Hib.

Amino Acid Sequence↗
PubMed1993

Identification of surface-exposed B-cell epitopes recognized by Haemophilus influenzae type b P1-specific monoclonal antibodies.

A panel of P1 synthetic peptides was synthesized to map the surface-exposed epitopes of Haemophilus influenzae type b outer membrane protein P1 recognized by three murine monoclonal antibodies (MAbs 7C8, 3E12, and 6B1). By using peptide-specific enzyme-linked immunosorbent assays, MAbs 6B1, 7C8, and 3E12 were shown to recognize distinct epitopes localized within residues 60 to 88, 165 to 193, and 400 to 437 of mature P1, respectively. Since MAb 7C8 was shown previously to be protective against certain H. influenzae type b subtypes in the infant rat model of bacteremia, its cognate epitope was further characterized by using truncated peptide analogs. Fine mapping of the 7C8 epitope by competitive inhibition studies revealed that it was localized within residues 184 and 193.

Amino Acid Sequence↗
PubMed1993

A major outer membrane protein of Moraxella catarrhalis is a target for antibodies that enhance pulmonary clearance of the pathogen in an animal model.

A murine immunoglobulin G monoclonal antibody (MAb) raised against outer membrane vesicles of Moraxella catarrhalis 035E was shown to bind to a surface-exposed epitope of a major outer membrane protein of this organism. This outer membrane protein, which had an apparent molecular weight of approximately 80,000 in sodium dodecyl sulfate-polyacrylamide gels, was designated CopB. MAb 10F3, reactive with CopB, bound to a majority (70%) of M. catarrhalis strains tested. More importantly, mice passively immunized with MAb 10F3 exhibited an enhanced ability to clear a bolus challenge of M. catarrhalis from their lungs, a result which suggested that CopB might have potential as a vaccine candidate. The M. catarrhalis gene encoding CopB was cloned in Escherichia coli, and nucleotide sequence analysis of the copB gene indicated that the CopB protein was synthesized with a leader peptide, a finding confirmed by N-terminal amino acid sequence analysis of the mature CopB protein purified from M. catarrhalis 035E. Southern blot analysis showed that chromosomal DNA from seven different M. catarrhalis strains hybridized with a probe comprising the majority of the copB structural gene from strain 035E. Additional data emphasizing the extent of conservation of the CopB protein among M. catarrhalis strains were obtained from Western immunoblot analyses with polyclonal antisera raised against CopB proteins from different M. catarrhalis strains used to probe the recombinant form of the CopB protein from strain 035E. The ability of the CopB protein to function as a target for biologically active antibodies and its apparent conservation among M. catarrhalis strains warrant further investigation of this outer membrane protein as a potential vaccine candidate.

Amino Acid Sequence↗
PubMed1993

Paradoxical effect of pilus expression on binding of antibodies by Haemophilus influenzae.

Haemophilus influenzae type b (Hib) pili are surface proteins that are associated with the ability of Hib to attach to human epithelial cells. Like pilus expression of other bacteria, expression of Hib pili undergoes phase variation. We observed that Hib in the piliated phase (Hib p+) bound monoclonal antibodies directed against six conserved, surface-exposed, nonpilus Hib outer membrane epitopes to a greater extent than Hib in the nonpiliated phase (Hib p-). However, after extended incubation, p+ and p- cells bound these antibodies in a similar fashion. The differential in nonpilus antibody binding to p+ and p- Hib was not related to the presence of the type b capsule. In addition, Hib p+ organisms whose pilin gene was insertionally inactivated and did not produce pili and Hib in the nonpiliated phase bound the nonpilus Hib antibodies similarly. Hib p+ and p- organisms did not differ in their binding of anti-type b capsule antibody, and the binding was specific for the epitopes recognized by the antibodies. In complement-dependent bactericidal assays, the nonpilus antibodies killed Hib p+ more effectively than Hib p-. The increased binding to, and killing of, Hib p+ by a variety of nonpilus antibodies may be important for host defense against invasive Hib.

Animals↗
PubMed1993

Reconstitution of a porin-deficient mutant of Haemophilus influenzae type b with a porin gene from nontypeable H. influenzae.

The major outer membrane protein (OmpP2) of nontypeable Haemophilus influenzae (NTHI) has been shown to vary markedly with respect to both size and the presence of specific surface-exposed epitopes among strains of this unencapsulated pathogen. In contrast, the OmpP2 proteins of H. influenzae type b (Hib) strains are well conserved at the level of primary protein structure and have in common several surface-exposed antigenic determinants that have not been detected in NTHI strains. The availability of an isogenic, avirulent Hib ompP2 mutant made it possible to investigate whether an NTHI OmpP2 protein could function properly in the Hib outer membrane. A plasmid shuttle vector (pGJB103) was used to clone the ompP2 gene from NTHI TN106 into a recombination-deficient H. influenzae strain in which expression of the NTHI OmpP2 protein was detected by means of an NTHI TN106 OmpP2-specific monoclonal antibody. The amino acid sequence of this NTHI OmpP2 protein, as deduced from the nucleotide sequence of the NTHI TN106 ompP2 gene, was determined to be 83% identical to that of the Hib OmpP2 protein. Transformation of this cloned NTHI ompP2 gene into the Hib ompP2 mutant yielded a Hib transformant strain that expressed the NTHI OmpP2 protein. Expression of this NTHI OmpP2 protein allowed the Hib ompP2 mutant, which normally grows poorly in vitro, to grow in a manner indistinguishable from that of the wild-type Hib strain. More importantly, the introduction of this functional NTHI ompP2 gene into the avirulent Hib ompP2 mutant restored the virulence of this strain to wild-type levels. These results indicate that an NTHI OmpP2 protein can be expressed and function properly in the Hib outer membrane.

Amino Acid Sequence↗
PubMed1992

Identification of a genetic locus of Haemophilus influenzae type b necessary for the binding and utilization of heme bound to human hemopexin.

The mechanism(s) used by Haemophilus influenzae to acquire the essential nutrient heme from its human host has not been elucidated. The heme carried by the high-affinity serum protein hemopexin is one potential source of this micronutrient in vivo. A colony-blot assay revealed that heme-human hemopexin-binding activity was shared among most capsular serotype b strains of H. influenzae but was uncommon among other strains. We have identified a recombinant clone binding heme-human hemopexin from a H. influenzae type b (Hib) genomic library expressed in Escherichia coli. Both the Hib strain and the heme-hemopexin-binding clone expressed a polypeptide of approximately 100 kDa that bound radiolabeled heme-hemopexin. Oligonucleotide linker insertion mutagenesis of the plasmid DNA from this recombinant clone was used to confirm that expression of the 100-kDa protein correlated with the heme-hemopexin-binding activity. Exchange of one of these mutant alleles into the Hib chromosome eliminated expression of both the 100-kDa protein and the heme-hemopexin-binding activity. Furthermore, this Hib mutant was unable to utilize heme-human hemopexin as a heme source.

Bacterial Proteins↗
PubMed1992

Animal models for the study of noninvasive Haemophilus influenzae disease: pulmonary clearance systems.

Nontypeable Haemophilus influenzae are increasingly being recognized as an important cause of disease in the lower respiratory tract. Information about the pathogenesis of these infections has been limited until recently by the paucity of experimental animal models for studying the host-parasite interaction in vivo. Three different animal models for investigation of the interaction of nontypeable H. influenzae with the lower respiratory tract have been used: Two involve the evaluation of short-term pulmonary clearance in mice or rats, while a new, long-term infection model in rats mimics the more chronic bronchopulmonary infections sometimes caused by these organisms. Results obtained in these three model systems indicate that both humoral and cellular immune mechanisms are involved in immunity to lower respiratory tract disease due to nontypeable H. influenzae.

Animals↗
PubMed1992

Virulent Treponema pallidum activates human vascular endothelial cells.

Perivascular lymphocytic infiltration, fibrin deposition, and endothelial cell abnormalities consistent with cellular activation are prominent histopathologic features of syphilis, a sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum. Because activated endothelial cells play important roles in lymphocyte homing and hemostasis, the ability of virulent T. pallidum to activate cultured human umbilical vein endothelial cells (HUVEC) was investigated. T. pallidum induced the expression of intercellular adhesion molecule-1 (ICAM-1) and procoagulant activity on the surface of HUVEC. Electron microscopy of T. pallidum-stimulated HUVEC revealed extensive networks of fibrin strands not observed in cultures without treponemes. ICAM-1 expression in HUVEC also was promoted by a 47-kDa integral membrane lipoprotein purified from T. pallidum, implicating a role for spirochete membrane lipoproteins in endothelial cell activation. The combined findings are consistent with the pathology of syphilis and provide the first evidence that a pathogenic spirochetal bacterium such as T. pallidum or its constituent integral membrane lipoprotein(s) can activate directly host vascular endothelium.

Antigens, CD↗
PubMed1992

Pulmonary clearance of Moraxella catarrhalis in an animal model.

The virulence mechanisms of Moraxella catarrhalis that are involved in producing pulmonary infection are unknown. A well-characterized murine model was used to study the pulmonary clearance of M. catarrhalis and analyze the histopathologic changes and the role of phagocytic cells in the infected lungs. Ten strains of M. catarrhalis from various isolation sites were evaluated for their ability to resist pulmonary clearance. The rates of clearance of these strains, based on the percentage of the original inoculum remaining at 6 h after challenge, varied considerably. Histopathologic examination of lungs infected with 2 strains that exhibited very different clearance rates revealed similar pathologic responses. Analysis of the phagocytic cell response to these 2 strains revealed significant alveolar recruitment of granulocytes at 3, 6, and 24 h after bacterial challenge. However, granulocyte recruitment in response to strain B22, which was cleared readily, was significantly greater than to strain 035E, which resisted pulmonary clearance. This model system should facilitate investigation of the molecular basis of the interaction between M. catarrhalis and the lower respiratory tract.

Animals↗
PubMed1992

The hbpA gene of Haemophilus influenzae type b encodes a heme-binding lipoprotein conserved among heme-dependent Haemophilus species.

A membrane-associated lipoprotein of Haemophilus influenzae type b has previously been shown to bind heme in vitro and to promote binding of this compound by Escherichia coli recombinants expressing this protein. The H. influenzae type b heme-binding protein A (HbpA) was found to be highly conserved with respect to both antigenicity and apparent molecular weight among heme-requiring Haemophilus species pathogenic for humans. To further the characterization of the structure and function of HbpA, the complete nucleotide sequence of its gene, hbpA, was determined. Analysis of the nucleotide sequence revealed a single large open reading frame of 1,638 bp encoding a protein of 546 amino acid residues, with a molecular weight of 60,695. The sequence of the amino-terminal end of this protein contained a potential site for lipid acylation and for cleavage by signal peptidase II, consistent with earlier biochemical evidence which indicated that HbpA is a lipoprotein. A search of GenBank for proteins with amino acid sequence similarity to HbpA revealed that the periplasmic dipeptide transport protein of E. coli, DppA, has 53% sequence identity to HbpA.

Amino Acid Sequence↗
PubMed1992

Use of electroporation to construct isogenic mutants of Haemophilus ducreyi.

Little is known about the genetics of Haemophilus ducreyi, the etiologic agent of chancroid. To develop a method for constructing isogenic mutants of this organism that could be utilized in pathogenesis-related studies, electroporation techniques were evaluated as a means of introducing DNA into this organism. Electroporation of the plasmid shuttle vector pLS88 into H. ducreyi yielded approximately 10(6) antibiotic-resistant transformants per microgram of plasmid DNA. Studies of the feasibility of moving mutated genes into H. ducreyi were initiated by using NotI linker insertion and mini-Tn10kan mutagenesis techniques to introduce insertion mutations into cloned H. ducreyi genes encoding cell envelope antigens. In the former case, a gene encoding chloramphenicol acetyltransferase was then inserted into the NotI linker site created in the cloned H. ducreyi gene. The recombinant Escherichia coli strains containing these mutated plasmids no longer expressed the homologous H. ducreyi cell envelope antigens, as evidenced by their lack of reactivity with monoclonal antibody probes for these H. ducreyi proteins. Subsequent electroporation of both circular and linearized forms of plasmids carrying these mutated H. ducreyi genes into the homologous wild-type strain of H. ducreyi yielded antibiotic-resistant transformants which also lacked reactivity with the cell envelope antigen-specific monoclonal antibodies. Southern blot analysis confirmed that homologous recombination had occurred in these monoclonal antibody-unreactive transformants, resulting in the replacement of the wild-type allele with the mutated allele. Allelic exchange was most efficient when linear DNA molecules were used for electroporation. These results indicate that electroporation methods can be utilized to construct isogenic mutants of H. ducreyi.

Blotting, Southern↗
PubMed1991

Endotoxin concentrations in cerebrospinal fluid correlate with clinical severity and neurologic outcome of Haemophilus influenzae type B meningitis.

Endotoxin concentrations were measured in paired samples of cerebrospinal fluid from 38 patients with Haemophilus influenzae type b meningitis. On admission, the median concentration of endotoxin in cerebrospinal fluid was 104 ng/mL and decreased rapidly in follow-up samples. From 17 to 48 hours after admission, 50% of the patients had concentrations of less than 1 ng/mL. Endotoxin concentrations correlated significantly with concentrations of interleukin 1 beta, protein, and glucose in cerebrospinal fluid, duration of secondary fever, and neurologic abnormalities during hospitalization and on follow-up examinations. Twenty-eight percent of patients with endotoxin concentrations of 100 ng/mL or more on admission had long-term complications, compared with none of those with lower endotoxin concentrations (relative risk, 2.31; 95% confidence interval, 1.53 to 3.48). These results indicate that quantitation of endotoxin in cerebrospinal fluid could be a valuable aid in identifying those children at increased risk of complications during Haemophilus influenzae type b meningitis and provide additional evidence that the Haemophilus influenzae type b meningitis lipo-oligosaccharide is important in the pathogenesis of meningitis.

Animals↗
PubMed1991

Blood-brain barrier alterations in bacterial meningitis: development of an in vitro model and observations on the effects of lipopolysaccharide.

To further examine the effects of purified Haemophilus influenzae type b lipopolysaccharide (LPS) on blood-brain barrier permeability, we have developed an in vitro model of the BBB. Microvascular endothelial cells were isolated from rat cerebral cortices by enzymatic digestion, dextran centrifugation, and separation on percoll gradients. The cells were determined to be endothelial in origin by positive fluorescent staining for Factor VIII-related antigen and the ability to take up acetylated low density lipoproteins, and their cerebral origin by the formation of junctional complexes in vitro. Cells were seeded onto semipermeable polycarbonate filters and permeability assessed by measuring traversal of radioactive albumin across the monolayer. Treatment of the cells with LPS at concentrations of 1.0 microgram/ml and 0.1 microgram/ml for 4 h led to statistically significant increases in albumin permeability of 4.6% (P = 0.001) and 5.6% (P less than 0.001), respectively, without evidence of cell death as assessed by release of lactate dehydrogenase into the media. These results indicate that LPS significantly increases albumin permeability across a monolayer of cerebral microvascular endothelial cells in the absence of host inflammatory cells. Future studies on the effects of LPS on intracellular regulation will determine the mechanisms responsible for these alterations.

Albumins↗
PubMed1991

Induction of suppurative arthritis in rabbits by Haemophilus endotoxin, tumor necrosis factor-alpha, and interleukin-1 beta.

Because Haemophilus influenzae type b (Hib) is the principal cause of suppurative arthritis in young children and its lipooligosaccharide (LOS) is thought to be the main virulence factor, Hib endotoxin was evaluated for its ability to induce synovial inflammation in rabbits. Also, the role of the cytokines tumor necrosis factor-alpha (TNF alpha) and interleukin-1 beta (IL-1 beta) in mediating the synovial inflammatory process was studied. Intraarticular inoculation of 2 pg to 20 ng of Hib LOS produced a dose-dependent increase in concentrations of leukocytes and protein in synovial lavage fluid that was significantly modulated by concomitant administration of rabbit TNF alpha- and rabbit IL-1 beta-specific antibodies. Inoculation of joints with either 10(4) IU of rabbit TNF alpha or 10 ng recombinant rabbit IL-beta induced synovial inflammatory changes similar to those observed after LOS intraarticular challenge. These data provide evidence for the role of Hib LOS in inducing suppurative arthritis and for the critical participation of TNF alpha and IL-1 beta in the initial events of the synovial inflammatory response.

Animals↗
PubMed1991

Detection of experimental Haemophilus influenzae type b bacteremia and endotoxemia by means of an immunolimulus assay.

The rapid quantitation of bacteria in blood was achieved by using a novel assay method for gram-negative bacterial lipopolysaccharide (endotoxin, LPS). The assay involves the capture of specific LPS onto microtiter plates by means of monoclonal antibodies directed against the oligosaccharide region of the LPS, followed by detection of the bound LPS by a chromogenic Limulus amebocyte lysate (LAL) system. This immunolimulus (IML) assay combines the specificity of monoclonal antibodies with the sensitivity of the LAL system to achieve the first specific, sensitive quantitation of bioactive endotoxin in plasma. In the animal model tested, Haemophilus influenzae type b (Hib) bacteremia in infant rats, there was a strong correlation between IML results and the concentration of Hib colony-forming units in blood samples (r = .845, P less than .001). Using antibodies with appropriate specificities, this approach should be useful for rapid detection of a wide range of gram-negative bacteria and endotoxins in blood.

Animals↗
PubMed1991

A temperature-dependent rabbit model for production of dermal lesions by Haemophilus ducreyi.

The fact that optimal in vitro growth of Haemophilus ducreyi occurs at 33 degrees C prompted evaluation of the effect of temperature on the ability of this organism to produce skin lesions in rabbits after intradermal inoculation. Animals housed at a reduced ambient temperature (15-17 degrees C) consistently developed necrotic lesions when injected intradermally with 10(5) cfu of H. ducreyi; this inoculum did not produce necrotic lesions in animals housed at normal room temperature (23-25 degrees C). Lesion production in this new model was dependent on both viability of the H. ducreyi inoculum and replication of these organisms after intradermal injection. Histopathologic examination of the lesions revealed that H. ducreyi infection of the rabbit dermis evolves from an acute inflammatory reaction to abscess formation. Evaluation of three additional strains of H. ducreyi in this model confirmed that lesion formation was not bacterial strain-dependent. This new temperature-dependent rabbit model for productive H. ducreyi infection will facilitate investigation of the molecular pathogenesis of chancroid.

Animals↗
PubMed1991

In vivo and in vitro expression of Haemophilus influenzae type b lipooligosaccharide epitopes.

An indirect immunofluorescence system involving monoclonal antibodies (MAbs) directed against surface epitopes of Haemophilus influenzae type b (Hib) lipooligosaccharide (LOS) was used to examine individual Hib cells in cerebrospinal fluid (CSF) from infants with Hib meningitis. In four of five CSF samples studied, 100% of the bacteria bound at least one LOS-directed MAb. When the bacteria from these CSF samples were grown in broth, most of these cells lost some or all of their ability to bind the MAb(s) that were bound by the same organisms present in human CSF. When in vitro-grown cells were used for intracisternal injection of rabbits, the populations of Hib cells observed in rabbit CSF after the development of meningitis generally resembled those of the respective broth-grown inocula in terms of their LOS antigenic characteristics. Hib cell populations recovered in infant rat CSF after intranasal challenge again had LOS antigenic characteristics similar to those of the in vitro-grown challenge inocula. These results indicate that a population of Hib cells growing in the infected human host may be quite different, with regard to its LOS antigenic characteristics, from the same Hib strain growing in vitro or in vivo in animal models.

Animals↗