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Biomedical subjects

E J Hansen

Publications and source records attributed to E J Hansen.

At least 91 records · Page 5Linked to original sources
PubMed1991

Molecular cloning of a gene involved in lipooligosaccharide biosynthesis and virulence expression by Haemophilus influenzae type B.

A wild-type Haemophilus influenzae type b (Hib) genomic DNA library was constructed in the plasmid shuttle vector pGJB103. A virulence-deficient lipooligosaccharide (LOS) mutant of Hib was used as a recipient for genetic transformation to screen this Hib genomic DNA library for genes involved in LOS expression. A recombinant plasmid containing a 7.8 kb PstI fragment of Hib DNA was shown to transform this LOS mutant to reactivity with a monoclonal antibody (mAb) specific for a wild-type LOS epitope. Transformation of two different virulence-deficient LOS mutants with a 4.4 kb BglII fragment of this recombinant plasmid yielded transformants which expressed LOS that bound the wild-type LOS-specific mAb and yielded profiles in sodium dodecyl sulphate/polyacrylamide gradient gel electrophoresis different from those of the original LOS mutants. These transformants with structurally altered LOS molecules also exhibited increased virulence in an animal model for invasive Hib disease. The virulence-transforming ability was further localized to a 1.8 kb BglII-AlwNI fragment of the Hib DNA insert. Nucleotide sequence analysis indicated the presence of a single large open reading frame within this fragment. This open reading frame contained 19 consecutive repeats of the tetramer CAAT near the 5' end. Linker insertion mutagenesis was used to demonstrate directly the involvement of this open reading frame in both LOS biosynthesis and virulence expression by Hib.

Amino Acid Sequence↗
PubMed1991

Molecular cloning, partial purification, and characterization of a haemin-binding lipoprotein from Haemophilus influenzae type b.

A library of genomic DNA fragments from Haemophilus influenzae type b (Hib) DL42 was constructed in plasmid pBR322, transformed into Escherichia coli strain RR1, and screened for recombinant clones with haemin-binding activity by plating onto haemin-containing agar. Expression of haemin-binding activity by clones correlated with the expression of a protein with an apparent molecular weight of 51,000 (51K) that was also recognized by anti-Hib strain DL42 serum in immunoblots. One recombinant clone, designated pHM2, with the smallest DNA insert (3.62 kb) was characterized further. Ethanol inhibition of expression of pHM2 in minicells revealed that the 51K protein was the result of a processing event involving a larger precursor. E. coli RR1(pHM2) adsorbed haemin in liquid suspensions as well as from solid media. Subcloning of a 2.6 kb fragment of pHM2 into a shuttle vector permitted the construction of a recombinant Hib clone, DL42(pHM1002), which overexpressed the 51K haemin-binding protein. This 51K protein appears to be peripherally associated with the inner, and possibly outer, membranes of Hib. Affinity chromatography on haemin-agarose was utilized to purify the haemin-binding protein from both E. coli RR1(pHM2) and Hib DL42(pHM1002) to near homogeneity. The use of the antibiotic globomycin in a minicell expression system and radioimmunoprecipitation analysis of Hib proteins intrinsically radiolabelled with [3H]-palmitate indicated that the 51K haemin-binding protein is a lipoprotein.

Anti-Bacterial Agents↗
PubMed1991

Enhanced attenuation of meningeal inflammation and brain edema by concomitant administration of anti-CD18 monoclonal antibodies and dexamethasone in experimental Haemophilus meningitis.

Antiinflammatory therapy has been shown to reduce the adverse pathophysiological consequences that occur in bacterial meningitis and to improve outcome from disease. In the present study, modulation of two principal steps of the meningeal inflammatory cascade was accomplished by concomitant administration of dexamethasone to diminish overproduction of cytokines in response to a bacterial stimulus and of a monoclonal antibody directed against adhesion-promoting receptors on leukocytes to inhibit recruitment of white blood cells into the subarachnoid space. Dexamethasone and antibody therapy produced a marked attenuation of all indices of meningeal inflammation and reduction of brain water accumulation after H. influenzae-induced meningitis in rabbits compared with results of each agent given alone and of untreated animals. In addition, the enhanced host's meningeal inflammatory reaction that follows antibiotic-induced bacterial lysis was profoundly ameliorated when dual therapy was administered without affecting clearance rates of bacteria from cerebrospinal fluid and vascular compartments. The combination of both therapeutic approaches may offer a promising mode of treatment to improve further the outcome from bacterial meningitis.

Animals↗
PubMed1990

Tumor necrosis factor alpha/cachectin and interleukin 1 beta initiate meningeal inflammation.

Although previous studies using human cytokines in rabbits and rats have provided evidence of the participation of tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) in the meningeal inflammatory cascade, the results obtained by several groups of investigators have been discordant or, at times, contradictory. In the present study, homologous cytokines were applied to the rabbit meningitis model. Intracisternal administration of 10(2)-10(5) IU of purified rabbit TNF-alpha (RaTNF-alpha) produced significant cerebrospinal fluid (CSF) inflammation. A similar response was observed after intracisternal inoculation of 5-200 ng of rabbit recombinant IL-1 beta (rrIL-1 beta). Preincubation of these two mediators with their specific antibodies resulted in an almost complete suppression of the CSF inflammatory response. In animals with Haemophilus influenzae type b lipooligosaccharide-induced meningitis, intracisternal administration of anti-rrIL-1 beta, anti-RaTNF-alpha, or both resulted in a significant modulation of meningeal inflammation. Simultaneous administration of 10(3) IU of RaTNF-alpha and 5 ng of rrIL-1 beta resulted in a synergistic inflammatory response manifested by a more rapid and significantly increased influx of white blood cells into the CSF compared with results after each cytokine given alone. These data provide evidence for a seminal role of TNF-alpha and IL-1 beta in the initial events of meningeal inflammation.

Animals↗
PubMed1990

Pulmonary host defenses and oropharyngeal pathogens.

The lower respiratory tract is repetitively inoculated with oropharyngeal bacteria and yet pneumonia is an infrequent event. Efficient mechanisms of antibacterial defense are present in the respiratory tract that eliminate microbes before their presence or multiplication leads to disease in the majority of instances. Resident pulmonary defenses consist of aerodynamic defenses, the mucociliary apparatus, alveolar macrophages, complement, and surfactant. These resident defenses can be augmented by the development of an inflammatory response or the development of specific immunity. Significant species variability exists in the efficiency and mechanisms of clearance for oropharyngeal organisms. Streptococci are cleared promptly, Branhamella catarrhalis is cleared slowly, whereas non-typable Haemophilus influenzae multiply before being cleared. A dual phagocytic system of alveolar macrophages and recruited polymorphonuclear leukocytes is required for clearance of most oropharyngeal microbes. Systemic immunization can significantly enhance clearance of non-typable H. influenzae, suggesting immunoprophylaxis might be possible for this organism.

Animals↗
PubMed1990

Characterization of a mutant of Haemophilus influenzae type b lacking the P2 major outer membrane protein.

An isogenic mutant of Haemophilus influenzae type b (Hib) lacking the ability to express the P2 major outer membrane (porin) protein was constructed and characterized in various model systems. Linker insertion mutagenesis of a cloned Hib DNA insert containing the P2 structural gene was used in conjunction with a genetic transformation system to obtain a transformant unreactive with a P2-specific monoclonal antibody. This transformant was shown to lack detectable P2 protein by both protein staining and immunoblot methods. The P2 mutant exhibited a generation time in complex broth medium that was significantly longer than that of the wild-type parent strain. The P2 mutant was also unable to produce detectable bacteremia in infant rats after intraperitoneal challenge, while the wild-type parent strain produced bacteremia in all animals challenged with this strain. Reintroduction of a wild-type copy of the P2 gene into this mutant yielded a transformant strain that had a generation time in vitro identical to that of the wild-type parent strain and that was also fully virulent in the infant rat model. These findings suggest that the ability to synthesize the P2 protein may be necessary but not sufficient for full expression of virulence by this pathogen.

Animals↗
PubMed1990

Multiple mechanisms in serum factor-induced resistance of Haemophilus influenzae type b to antibody.

Incubation of Haemophilus influenzae type b at less than or equal to 10(7) CFU/ml with serum ultrafiltrate induces a phenotypic conversion in which complement-mediated bactericidal activity by somatic antibodies decreases while killing by capsular antibody is unchanged. Conversion had been shown to occur in a capsule-deficient (b-) mutant of strain Eag (thus appearing independent of capsulation), to include an increase in lipopolysaccharide content, and to be inhibited by chloramphenicol or puromycin. In the present study, in several strains not previously examined, conversion was not inhibited by the drugs and the corresponding b- mutants did not convert. Incubation in ultrafiltrate was also found to increase capsulation, as detected by radioassay, only 1.6-fold in Eag but 4.5-fold in DL26, the strain with the largest increase in resistance; moreover, complement-mediated opsonization by capsular antibody was greatly decreased. Thus, multiple mechanisms, capsule dependent as well as independent, appear to contribute to the serum factor-induced resistance of H. influenzae type b to antibody.

Antibodies, Bacterial↗
PubMed1990

Effect of mutations in lipooligosaccharide biosynthesis genes on virulence of Haemophilus influenzae type b.

Chemical mutagenesis techniques and genetic transformation methods were used to construct isogenic mutants of Haemophilus influenzae type b (Hib) defective in the ability to synthesize lipooligosaccharide (LOS). A mutant (17B) which expressed a LOS molecule with an altered oligosaccharide was less virulent than the wild-type parent strain, as determined by measurement of the ability of these strains to produce bacteremia in infant rats after intranasal challenge. Further mutagenesis of this mutant strain yielded two new mutants with different LOS phenotypes. Mutant 7A was slightly sensitive to the bactericidal activity present in normal infant rat serum and was even less virulent than its immediate parent strain (17B) in the intranasal challenge model. However, both mutants 17B and 7A could produce bacteremia and meningitis when introduced into infant rats by the intraperitoneal route. The other LOS mutant (14A) derived from mutant 17B exhibited a level of virulence equivalent to that of the original wild-type strain. Genetic transformation of wild-type chromosomal DNA into the essentially avirulent mutant 7A and selection of transformants on the basis of their LOS antigenic characteristics resulted in the sequential restoration of full virulence to this mutant. These findings suggest that LOS is involved on at least two different levels in the ability of Hib to produce invasive disease in the infant rat model. Changes in LOS phenotype can independently affect the ability of Hib to produce bacteremia after intranasal challenge and the sensitivity of Hib to killing by normal infant rat serum. These results reinforce the significance of Hib LOS in the expression of virulence by this pathogen.

Administration, Intranasal↗
PubMed1990

Specific detection of Haemophilus influenzae type b lipooligosaccharide by immunoassay.

Three monoclonal antibody (MAb)-based immunoassays were developed for specific detection of Haemophilus influenzae type b (Hib) lipooligosaccharide (LOS). (i) Hib LOS was captured onto microtiter plates by polyclonal Hib-directed antibodies and detected with MAbs to the oligosaccharide component of Hib LOS in an enzyme-linked immunosorbent assay, (ii) The high affinity of polymyxin B for lipid A was used to bind Hib LOS to microtiter wells, and the oligosaccharide-specific MAbs were used as the detection system in the polymyxin B-MAb assay. (iii) Hib LOS solubilized in detergent was captured by MAbs, and the immobilized LOS was detected with a chromogenic Limulus amebocyte lysate method in the immunolimulus assay. Endotoxin concentrations were measured in in vitro samples and cerebrospinal fluid samples from rabbits with experimental Hib meningitis. The results were compared with those obtained with the standard chromogenic Limulus amebocyte lysate assay. There were significant correlations between the results of all four assays. These new immunoassays provide methods for specific detection of Hib LOS in laboratory fluids and in research involving quantification of Hib endotoxin in experimental animal models.

Animals↗
PubMed1989

Specific detection of Haemophilus influenzae type b lipooligosaccharide by a polymyxin B monoclonal antibody assay.

A monoclonal antibody (MAb)-based enzyme immunoassay was developed for detection of Haemophilus influenzae type b (Hib) lipooligosaccharides (LOS). The high affinity of polymyxin B for lipid A was used to bind the Hib LOS to microtiter wells. The immobilized LOS was detected with MAbs directed against the oligosaccharide component of Hib endotoxin. Hib LOS concentrations were measured in in vitro samples and in cerebrospinal fluid (CSF) sample obtained from rabbits with experimental Hib meningitis. The sensitivity of the assay was 1 ng LOS/ml sample and the results obtained with this assay correlated significantly with those obtained with the standard Limulus amebocyte lysate assay. This new assay provides a method for specific detection of Hib LOS in CSF samples and in aqueous laboratory fluids. This general methodology should also be useful for experimental research involving specific LPS/LOS molecules.

Animals↗
PubMed1989

Induction of meningeal inflammation by outer membrane vesicles of Haemophilus influenzae type b.

Haemophilus influenzae type b (Hib) lipooligosaccharide (LOS) induces meningitis in an established rabbit model of experimental meningitis. To evaluate the inflammatory ability of Hib LOS in its native state as part of outer membrane of Hib, the CSF inflammatory response to Hib outer membrane vesicles (OMV) was compared with that produced by Hib LOS. A significant dose-dependent meningeal inflammatory response, as evidence by increased concentrations of white blood cells and protein and lactate concentration in CSF, was found after intracisternal injection of Hib OMV containing 0.02-200 ng of LOS. On a LOS weight basis, purified LOS and LOS in OMV did not differ significantly with regard to the CSF inflammatory response. Preincubation of Hib OMV with monoclonal antibodies directed against cell surface-exposed epitopes on Hib LOS did not affect the inflammatory ability of OMV, whereas preincubation of OMV with polymyxin B significantly reduced both pleocytosis and lactate concentration. Thus, Hib OMV represents a relevant nonreplicating vehicle in which Hib LOS might interact with CNS tissues to produce meningitis.

Analysis of Variance↗
PubMed1989

Modulation of inflammation and cachectin activity in relation to treatment of experimental Hemophilus influenzae type b meningitis.

By use of an experimental lapin model, the effect of ceftriaxone therapy on inflammation during Hemophilus influenzae type b (Hib) meningitis was evaluated. Meningitis was induced by intracisternal inoculation of 2 x 10(4) to 2 x 10(5) colony-forming units of Hib. Administration of ceftriaxone 6 h after infection provoked rapid bacterial lysis associated with greatly increased concentrations of bacteria-free endotoxin (lipooligosaccharide) and tumor necrosis factor alpha (TNF) in the cerebrospinal fluid (CSF). CSF TNF activity peaked 2 h after initiation of antibiotic therapy (24.4 +/- 2 ng/ml), was significantly higher than that in untreated controls (1.4 +/- 1.1 ng/ml; P less than .05), and was associated with a substantially increased inflammatory response as reflected by higher CSF white blood cell count (24,500 +/- 8,151 vs. 1,920 +/- 644 in untreated controls; P less than .05), lower glucose, and higher protein and lactate concentrations. Simultaneous administration of dexamethasone with antibiotic therapy resulted in a significant reduction in CSF TNF activity that was associated with a substantial reduction in CSF white blood cell count. These data suggest that initiation of ceftriaxone therapy in experimental Hib meningitis exacerbates the meningeal inflammatory response, which can be modulated by concurrent dexamethasone administration.

Animals↗