Opsono-phagocytosis of non-encapsulated Haemophilus influenzae.
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Enzyme-Linked Immunosorbent Assay↗
Biomedical subjects
L Vogel
Publications and source records attributed to L Vogel.
Interferon-gamma downregulates the proliferative response of hapten-specific B cells stimulated by antigen and cytokines.
IFN-gamma plays a role in many aspects of cellular interactions, both positive and negative. Among its functions during the immune response, the antagonistic effects of IFN-gamma and IL-4 are well documented. Observations in our laboratory suggested that IFN-gamma could also interfere with the activation of single, antigen-specific B cells by antigen and other cytokines. Closer examination revealed that IFN-gamma reduced the number of proliferating cell clones in response to antigen and a variety of cytokines, alone or in combination. Cell viability remained at the initial level and the cells were still able to produce Ig, albeit to a lesser extent than in the absence of IFN-gamma. On the other hand, the frequency of IgM secreting clones was not affected, whereas the total amount of secreted IgM was lower in the presence of IFN-gamma, probably due to the reduced cell number and a decrease in Ig production. In addition, proliferation was prevented when B cells were pre-incubated with IFN-gamma and then stimulated by other cytokines. Kinetic studies revealed that INF-gamma had to be present from the onset of culture because delayed addition did not inhibit the proliferation of the B cells. After its initial action, IFN-gamma could be removed without abolishing the negative signal for proliferation. From these results it can be concluded that IFN-gamma transmits a signal that causes B cells to stop proliferating and prevents them from forming large clones.
Animals↗
Does environmental ethics need a metaphysical grounding?
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Bioethics↗
The composition of the intracellular domains of the B cell antigen receptor complex studied from the cytoplasmic side of the membrane.
Immunoglobulins of the classes M and D function as antigen receptors on B lymphocytes. They are linked to other proteins to form B cell antigen receptor (BCR) complexes which transduce the signal triggered by the binding of antigen. In order to study the components that interact with BCR complexes in the cell it is essential that they are accessible to biochemical studies. Therefore, we have developed a simple and rapid method that allows the purification and labelling of B lymphocyte plasma membranes. For this, B cells are attached to polyacrylamide beads. Upon disruption of the cells, bead-bound membranes are obtained which expose the cytoplasmic side into the medium. The membrane proteins can then be radioiodinated and eluted with detergents. The combination of the improved methods for the preparation of bead-attached membrane patches and radiolabelling of the proteins has allowed for the first time an investigation into the cytoplasmic side of the BCR complex. All the subunits that had been previously described could be detected in 2D autoradiographs. Furthermore, it could be shown that the protein Ig-beta, which is part of an Ig-associated heterodimer, is predominantly labelled at the extracellular domain. The second component, Ig-alpha, is labelled to a higher degree at its intracellular domain. In addition, further proteins could be detected exclusively at the cytoplasmic side of the membrane. Results from 2D autoradiographs show that they may form heterodimers. These proteins are candidates for the interaction of BCR complexes with further members of the signalling cascade, such as protein tyrosine kinases and/or G proteins.
Animals↗
Quantitative flow cytometric analysis of opsonophagocytosis and killing of nonencapsulated Haemophilus influenzae by human polymorphonuclear leukocytes.
Since nonencapsulated Haemophilus influenzae persists in the lower respiratory tracts of patients with chronic bronchitis despite the presence of specific antibodies, complement, and polymorphonuclear leukocytes (PMNs), opsonophagocytosis of H. influenzae was analyzed. Nonencapsulated H. influenzae isolated from the sputa of chronic bronchitis patients was labeled with fluorescein isothiocyanate and incubated with human PMNs in the presence of complement and antibodies for 30 min at 37 degrees C. Candida albicans was added to each sample as an internal standard, and the reduction of the number of bacteria was determined by flow cytometry. Fluorescence quenching with ethidium bromide was used to discriminate between intracellular and extracellular bacteria. Opsonophagocytosis of viable H. influenzae d1 was 17% +/- 29% in the presence of complement and human pooled sera containing high titers of strain-specific antibodies. Opsonophagocytosis of six other H. influenzae strains was also poor. Under the same conditions, opsonophagocytosis of Staphylococcus aureus was 90% +/- 5%, and opsonophagocytosis of C. albicans was 55% +/- 23%. About half of the number of H. influenzae bacteria associated with PMNs was internalized. Opsonophagocytosis of heat-killed H. influenzae d1 (41% +/- 20%) was higher than that of viable bacteria of the same strain (P < 0.05). This result suggests that the accessibility of epitopes on H. influenzae for opsonizing antibodies is better on killed than on viable bacteria. We conclude that viable nonencapsulated H. influenzae is poorly opsonophagocytized in the presence of strain-specific antibodies and complement.
Bacterial Capsules↗
Opsonic antibodies to outer membrane protein P2 of nonencapsulated Haemophilus influenza are strain specific.
The ability of monoclonal antibodies (MAbs) specific for variable and conserved epitopes of outer membrane protein (OMP) P2 (b,c) of nonencapsulated Haemophilus influenza to promote opsonophagocytosis of this bacterium by human polymorphonuclear leucocytes (PMNs) was determined by flow cytometry. MAbs rendering PMNs fluorescent because of association with fluorescein isothiocyanate-labelled bacteria were defined as stimulating opsonophagocytosis. Opsonophagocytosis was dependent on the presence of both antibodies and complement. Of the 14 MAbs directed to the variable parts of OMP P2 (L. van Alphen, P. Eijk, L. Geelen-van den Broek, and J. Dankert, Infect. Immun. 59:247-252, 1991), 9 stimulated opsonophagocytosis. Four of the five nonopsonophagocytic MAbs that were immunoglobulin G1 were unable to cause complement activation. The MAbs promoting opsonophagocytosis included MAbs specific for one or more OMP P2 antigenic variants of H. influenzae strains isolated from patients with chronic bronchitis during persistent infection. MAbs cross-reacting in enzyme-linked immunosorbent assays with nonrelated H. influenzae did not promote opsonophagocytosis of strains from other patients. Opsonophagocytosis was not observed in the presence of three MAbs reacting with OMP P2 epitopes common in H. influenzae. These results indicate that OMP P2-dependent opsonophagocytosis of nonencapsulated H. influenzae is strictly strain specific.
Animals↗
New York University therapeutic roundtable: a panel of experts answer questions on the treatment of challenging cases.
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Adult↗
Effects of combinations of cytokines on murine antigen-specific B lymphocytes.
Cytokines are among the most important mediators in the immune response. They act on B lymphocytes at various stages along the activation pathway. To study the effects of combinations of cytokines, we have used an antigen-specific single B cell-cloning system devised in this laboratory. We report here that IL-1 can enhance the proliferative response and IgM secretion by B cells induced by IL-6. The results of a similar study demonstrated that IL-1 can also enhance the effects of IL-5 on B cell proliferation and IgM secretion in an additive manner. Kinetic analyses showed that IL-1 had to be present from the beginning of the culture for an optimum cooperative effect, whereas the addition of IL-6 could be delayed up to 2 days without a significant reduction of the response. In contrast, IL-5 had to be added together with IL-1 at the onset of culture to promote an optimum response. The responses elicited by IL-1 plus IL-6, IL-1 plus IL-5, and IL-4 plus IL-5 were almost identical. The addition of further cytokines to these cultures gave no enhancement above the effects observed with the two-cytokine combinations.
Animals↗
Changes in the expression of Ig-associated proteins on B lymphocytes activated by anti-IgM antibodies.
Binding of antigen to receptor complexes on B cells elicits a cascade of intracellular signalling events leading to proliferation and, together with T-cell help, Ig secretion. Components of the antigen receptor (AgR) complex have been demonstrated to be either covalently bound or associated with surface Ig (sIg) molecules. The function of these proteins is still unknown. In order to address this question, we have stimulated B cells with anti-mu antibodies and have studied possible changes in the expression of AgR complexes. After anti-mu stimulation, the IgM molecules disappeared rapidly from the cell surface together with the covalently bound proteins. The IgM molecules were internalized and probably degraded. The IgM-associated heterodimer Ig-alpha/Ig-beta was also removed from the cells, leaving the IgD-associated heterodimer unaffected. Two proteins showed an enhanced association with sIg after 15 min and then were gradually removed from the cell surface. Two other proteins became increasingly attached to sIg. This association remained stable for the rest of the culture period (up to 4 h). Further studies are underway to characterize these proteins more closely and to examine possible interactions with downstream members of the signalling cascade.
Animals↗
Immunological properties of recombinant porin of Haemophilus influenzae type b expressed in Bacillus subtilis.
The major surface-located, channel-forming protein in the outer membrane of Haemophilus influenzae type b (Hib) is porin (341 amino acids; M(r), 37,782). In order to generate Hib porin that is devoid of lipooligosaccharides and capsular polysaccharide, the Hib porin gene ompP2 was subcloned into a plasmid vector and recombinant Hib porin was expressed in Bacillus subtilis. Recombinant porin was produced in large quantities in B. subtilis and formed intracellular inclusion bodies. Recombinant porin was extracted from inclusion bodies and shown to be active in forming pores in synthetic black lipid membranes. However, these pores demonstrated different pore characteristics than wild-type Hib porin. Mouse hyperimmune sera against recombinant porin were generated and subjected to epitope scanning with a library of 336 overlapping synthetic hexapeptides that corresponded to the entire sequence of Hib porin. The epitope specificities of the anti-recombinant porin antibodies were similar to those of antibodies against Hib porin: selected regions near the amino terminus which include a buried loop in the native structure of Hib porin were more immunogenic than regions at the carboxy terminus. Although some mouse anti-recombinant porin antibodies mediated complement-dependent binding to Hib by polymorphonuclear leucocytes in opsonophagocytosis assays, the antibodies were not bactericidal, nor did they abrogate bacteremia in the infant rat model of infection. It was concluded that the native state of Hib porin is required for the generation of a protective immune response against the bacterium.
Animals↗
Prevalence of eating disorders in three Cambridge general practices: hidden and conspicuous morbidity.
The aims of this study were to investigate the prevalence of anorexia nervosa, bulimia nervosa and partial syndromes in women general practice attenders to establish the relative proportions of 'conspicuous' and 'hidden' morbidity. A consecutive series of 540 women patients aged 16-35 years attending their family doctor were screened using a specially devised questionnaire, the weight and dietary practices survey. A total of 115 patients were selected for further assessment and of these 101 patients were interviewed using a standardized diagnostic interview for DSM III-R eating disorders. The prevalence of anorexia nervosa was 0.2% (one case), of bulimia nervosa 1.5% (eight cases) and of partial syndrome bulimia nervosa 5.4% (29 cases). Half of the cases of bulimia nervosa had not been identified by the general practitioner and two of these patients had been referred to specialists for treatment of secondary complications of the eating disorder. Hidden cases of bulimia nervosa or partial syndromes are relatively common in general practice. Certain key questions could be used by general practitioners in order to identify women with eating disorders.
Adolescent↗
Hydrophobic labelling of membrane-embedded proteins with lipophilic reagents. Incorporation of [125I]INA and [125I]TID into B lymphocyte membrane immunoglobulins.
Hydrophobic labelling is frequently used in the study of membrane-inserted domains of intrinsic proteins. However, the published procedures, fail to incorporate sufficient radioactivity into membrane immunoglobulins of B lymphocytes to permit investigation of their subunit structures and associations with other proteins. In order to increase the specific radioactivity of [125I]iodonaphthylazide ([125I]INA), an improved method for the synthesis of the reagent was developed. In addition, the optimal conditions for labelling B lymphocytes with [125I]INA and the commercially available reagent 3-(trifluoromethyl)-3-(3'-[125I]iodophenyl)diazirine ([125I]TID) were elaborated. Under these optimized conditions, Ig molecules labelled with [125I]INA and [125I]TID were isolated and analysed in detail by SDS-PAGE. The usefulness of the two reagents for the investigation of lipid-embedded domains of membrane proteins is discussed.
Animals↗
Immunoglobulin subunits of murine B lymphocytes: structure and associations with other membrane proteins.
The Ig subunit structure of murine B lymphocytes was studied by employing different radiolabelling techniques in combination with chemical cross-linking. The main membrane structure of IgM was a half molecule that was disulphide-linked to proteins with MW 30,000, 45,000 and 55,000, respectively. Small amounts of mu 2L2, microL disulphide-linked to a protein with MW 50,000, and free microL were also detected. The main IgD structures were half molecules disulphide-linked to two proteins with MW 14,000 and two proteins with MW 16,000. Furthermore, IgD half molecules disulphide-linked to a protein with MW 16,000 and free half molecules could be demonstrated. Labelling with hydrophobic reagents showed that all Ig molecules and the protein with MW 50,000, linked to microL, penetrated the lipid bilayer, whereas the other IgM- and IgD-linked proteins probably did not. Additional proteins which were associated exclusively with IgM were detected by chemical cross-linking. These findings offer new possibilities for the investigation of the function(s) of antigen receptors on B cells.
Animals↗
Infections due to gentamicin-resistant Staphylococcus aureus strain in a nursery for neonatal infants.
An apparently homogeneous strain of Staphylococcus aureus resistant to gentamicin (Gmr), kanamycin, tobramycin, and sisomicin, but susceptible to amikacin and netilmicin, caused multiple infections in neonatal infants in a special care nursery. Nasal cultures revealed a high rate of carriage of the Gmr staphylococcus in infants without clinical infection. Segregating patients according to a modified cohort system and use of careful aseptic techniques led to apparent elimination of the Gmr strain. The resistance to aminoglycosides in this strain was mediated by an aminoglycoside 6'-N-acetyltransferase and a gentamicin phosphotransferase. Genetic determinants for these enzymes were borne on a circular covalently closed plasmid of approximately 11 megadaltons. These resistance determinants closely resemble those found in isolates of S. aureus that have caused nosocomial infections in patients in Europe.
Gentamicins↗
Chicken embryo model for type III group B beta-hemolytic streptococcal septicemia.
A lethal septicemia was induced in 11- and 12-day-old chicken embryos with intravenous inoculation of relatively small numbers of a clinical isolate (GBBHS-III-Bell) or a reference strain (GBBHS-III-D136-C) of group B beta-hemolytic streptococci (GBBHS). GBBHS-III-Bell was more virulent than GBBHS-III-D136-C, and 11-day-old chicken embryos were more susceptible than 12-day-old chicken embryos. Type-specific rabbit antisera protected the embryos from bacterial challenge, and this protective effect was absorbed with homologous but not heterologous GBBHS strains. A heterologous antiserum and normal rabbit sera provided some protection, which could be absorbed with either homologous or heterologous GBBHS strains. The chicken embryo is a suitable animal model for the study of infection and immunity with GBBHS type III.
Animals↗
Neurological assessment of the child with head trauma.
Dental trauma is a type of head trauma. It is essential that the dentist be able to assess the gross neurological status of the child presenting with head injury and to recognize acute and delayed signs of nerve injury. Time does not permit the dentist a leisurely review of necessary skills at the time of an emergency visit. He or she must be prepared in advance with a consistent and simple approach for the management of head trauma in children. Time of entry into care is critical to the prognosis.
Child↗