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Biomedical subjects

G Pluschke

Publications and source records attributed to G Pluschke.

At least 55 records · Page 3Linked to original sources
PubMed1989

Use of hybridoma immunoglobulin switch variants in the analysis of the protective properties of anti-lipopolysaccharide antibodies in Escherichia coli K1 infection.

Functional properties of rat immunoglobulins obtained from hybridoma isotype switch variants were studied in vivo in a rat model for neonatal bacterial sepsis. Escherichia coli 018:K1, a common cause of human neonatal sepsis and meningitis, was injected intravenously into 6-day-old rats after incubation with 018-specific antibodies IgM, IgG1, IgG2a, IgG2b, IgG2c, IgE and IgA. The clearance of bacteria treated with saline or IgE was low, whereas monoclonal antibodies of other isotypes triggered hepatic sequestration and killing of the K1 E. coli cells. All four IgG subclasses were more efficient than IgM and IgA. Comparable results were obtained upon injecting antibodies into rats with an established fulminating bacteraemia. IgM was inactive in animals depleted of complement with cobra-venom factor (CVF), whereas IgG2b was able to trigger hepatic clearance independently of complement.

Animals↗
PubMed1988

Host factors in the resistance of newborn mice to K1 Escherichia coli infection.

It is not clear which factors are responsible for the deficient resistance of human neonates to K1 E. coli sepsis and meningitis. To evaluate the relative importance of different defense mechanisms against bacterial invasion, we have analyzed the sensitivity of newborn mice with known immune deficiencies to infection after oral challenge with virulent K1 E. coli. T and B lymphocyte and complement (C5) defects had no significant effect on natural resistance. In contrast, both endotoxin-hyporesponsive mouse strains tested were highly sensitive. This susceptibility to infection was strongly age dependent. Infant endotoxin-hyporesponsive mice were killed by i.p. injection of less than ten virulent K1 E. coli cells. In contrast, endotoxin-responsive animals and F1 hybrids derived from crosses between endotoxin-responsive and hyporesponsive mice survived an injection with up to 10(4) bacteria. Mutants of a virulent 018:K1 E. coli strain defective in the synthesis of the capsular polysaccharide or the O-antigen of lipopolysaccharide were avirulent as were 01:K1 bacteria, which are under-represented among E. coli isolates from neonatal meningitis. Endotoxin-hyporesponsive mice were protected from lethal bacterial challenge by monoclonal IgG specific for the O-antigen of the challenge strain or by human recombinant interleukin 1. A fulminant bacterial multiplication in the bloodstream of endotoxin-hyporesponsive mice was observed after i.v. injection of 100 virulent K1 E. coli cells. Persistent bacteremia with 10(5) to 10(6) bacteria per ml of blood resulted in death of the animals one to two days after challenge. In the bloodstream of endotoxin-responsive mice the bacteria proliferated to a comparable extent within the first 6 h after challenge. Thereafter they were rapidly cleared from the circulation and the animals recovered from the infection.

Animals↗
PubMed1987

Isolation of rat immunoglobulin class switch variants of rat-mouse hybridomas by enzyme-linked immunosorbent assay and sequential sublining.

The use of sequential sublining in combination with highly specific and sensitive enzyme-linked immunosorbent assays for the isolation of spontaneous rat Ig heavy chain class switch variants is described. These methods allowed us to isolate switch variants from mouse-rat hybridoma lines secreting monoclonal rat antibodies. Switch variants from IgM to IgG2a, from IgG2a or IgG2b to IgE and from IgE to IgA were obtained. Members of the BA1.2 family, which consists of IgG2b, IgE and IgA antibodies are shown to exhibit identical rhamnose-inhibitable binding to the O18A antigen of Escherichia coli and to the paratope-associated anti-idiotypic antibody BA114.

Animals↗
PubMed1986

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and monoclonal antibodies as tools for the subgrouping of Escherichia coli lipopolysaccharide O18 and O23 antigens.

The lipopolysaccharide (LPS) of Escherichia coli O18 isolated from a wide variety of sources was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Four different LPS types, designated O18A, O18A1, O18B, and O18B1, were identified. Most O18 strains possess O18A, O18A1, or O18B LPS types, and these types are clonally associated. A reference test strain with the classical O18ab designation possessed O18B LPS, while two reference O18ac strains possessed O18A and O18A1 LPS, respectively. A panel of 15 anti-O18A B-cell hybridomas was isolated. Enzyme-linked immunosorbent assays revealed that some of the monoclonal antibodies produced by these cells recognize different epitopes. Four of these antibodies suffice to distinguish the four O18 types. Numerous strains whose LPS had been typed by SDS-PAGE were tested by agglutination with seven monoclonal antibodies whose specificities had been determined by enzyme-linked immunosorbent assays. The results indicated a perfect correlation between the two methods. Rabbit antisera raised against O18A bacteria agglutinated boiled bacteria of each of the O18 LPS types efficiently. The antisera were adsorbed with bacteria possessing each of the LPS types. The adsorbed sera only distinguished between two groups: O18A and O18A1 versus O18B and O18B1, as shown by agglutination assays and Western blotting. E. coli O4 and O23 and Serratia marcescens O8 antigens, which are reputed to cross-react with O18, were also analyzed. One O4, one O8, and four O23 strains were tested. All made an LPS which was distinguishable from O18 LPS types by SDS-PAGE. Each O23 strain synthesized a different LPS, and three of them synthesized only few short chains. Some of the monoclonal antibodies reacted with O4, O8, and O23A LPSs. The results are interpreted as indicating that numerous E. coli O serogroups will prove to be chemically heterogeneous and that future analyses of subgroup heterogeneity should be guided by results from SDS-PAGE and rely preferentially on monoclonal antibodies as opposed to rabbit hyperimmune sera.

Antibodies, Bacterial↗
PubMed1986

A reexamination of the O1 lipopolysaccharide antigen group of Escherichia coli.

A total of 64 Escherichia coli strains of the O1 serogroup were tested for the migration pattern of their lipopolysaccharides (LPS) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. O1:K1 and O1:K51 strains of the OMP5 outer membrane protein pattern possessed LPS with a doublet pattern (O1A1) or the lowermost band of the O1A1 doublet (O1A2). O1:K1 strains of the OMP9 pattern possessed LPS referred to as O1A, which corresponded to the uppermost band of the O1A1 doublet pattern. A few O1:K? strains possessed LPS of different migration patterns (O1B and O1C). O1A and O1A1 LPS were indistinguishable by chemical techniques, and both reacted with each of 10 different monoclonal antibodies tested. However, O1A1 had an additional epitope within the additional band in each doublet, as demonstrated by adsorption experiments with hyperimmune rabbit sera followed by Western blotting. Furthermore, purified polysaccharide from O1A bacteria was incapable of inhibition in enzyme-linked immunosorbent assays performed with O1A1 LPS as antigen and adsorbed, specific anti-O1A1 antibodies, whereas O1A1 polysaccharide inhibited this reaction. O1B and O1C LPS differed in all respects tested, including chemical composition, from O1A and O1A1 LPS.

Animals↗
PubMed1986

Complete two-dimensional gel electrophoresis pattern of de novo synthesized acute phase reactants.

The early stages of inflammatory responses are characterized by the rapid synthesis of a heterogenous group of plasma proteins known as acute phase reactants. We show that the complex changes in the serum protein composition of mice in response to infections can easily be analysed by two-dimensional (2D) gel electrophoresis, which allows simultaneous analysis of numerous proteins from small volume samples. While changes in the protein composition can be visualized by silver staining, radiofluorography of gels loaded with serum samples from mice that had been labelled in vivo with 35S-methionine allows an analysis of the changes in the pattern of protein synthesis. Thus, these techniques should allow the evaluation of the relative contributions of alterations of protein synthesis and catabolism to the changes in the overall concentration of individual acute phase reactants. Identification of proteins in the 2 D gel pattern can be easily accomplished by co-electrophoresing small serum samples together with immunoprecipitates obtained from in-vivo labelled serum. Using this approach we were able to identify some of the major acute phase reactants of mice. Some of these proteins, like haptoglobin and haemopexin, show concentration increases that are characteristic for type III reactants like C-reactive protein (CRP) or serum amyloid A component (SAA) in man. Results obtained with serum from healthy and infected human newborns indicate that 2D gel electrophoresis could be used to analyse changes in human plasma protein profiles, which would make it a valuable tool for diagnosis and management in certain clinical situations.

Acute-Phase Proteins↗
PubMed1985

Antibodies to O-antigen of lipopolysaccharide are protective against neonatal infection with Escherichia coli K1.

Monoclonal IgM specific for the O18 antigen conferred passive protection to 1-week-old rats against bacteremia and killing after oral challenge with O18:K1 Escherichia coli. Specific protection of the pups was also achieved by immunizing the pregnant rats with purified O18 lipopolysaccharide. We suppose that most human newborns that are colonized by potentially invasive K1 E. coli are protected by the transplacental transfer of anti-lipopolysaccharide immunoglobulin G, and we suggest that treatment with such antibodies might in the future be considered a therapeutic option. Rat serum from 1-week-old animals had only about one-third of the complement hemolytic activity of adult rat serum. This low level of hemolytic activity correlated with a relatively poor bactericidal activity in antibody-dependent and antibody-independent bactericidal in vitro assays. Monoclonal anti-O18 immunoglobulin M, although protective in vivo and bactericidal when added to adult rat serum, only poorly inhibited the multiplication of O18:K1 cells in serum from 1-week-old rats. This suggests that other elements of host defense besides complement participate in antibody-mediated in vivo protection.

Agglutination Tests↗
PubMed1984

Lipopolysaccharide, capsule, and fimbriae as virulence factors among O1, O7, O16, O18, or O75 and K1, K5, or K100 Escherichia coli.

K1, K5, and K100 Escherichia coli isolates of the lipopolysaccharide antigen types O1, O7, O16, O18, or O75, which had formerly been assigned to clonal groupings were compared with K? E. coli isolates and with laboratory-derived mutants defective in capsule or lipopolysaccharide synthesis. The amount of K1 capsule, the length distribution of the lipopolysaccharide, and the expression of type I and P fimbriae were determined. The clonal groupings were uniform with regard to these properties within each group but different from each other. Many of the K? strains differed from the clonal representatives. The results are interpreted with regard to the different diseases caused by each of these bacterial groups.

Cell Membrane↗
PubMed1984

Degree of antibody-independent activation of the classical complement pathway by K1 Escherichia coli differs with O antigen type and correlates with virulence of meningitis in newborns.

A total of 95 K1 Escherichia coli strains of the O (lipopolysaccharide) serotypes O1, O7, or O18 had been analyzed previously for the ability to cause bacteremia after colonizing the gut of newborn rats. In this study, these strains were tested for their resistance to the bactericidal activity of rat serum. All strains that had caused bacteremia in a high percentage of the inoculated rats were able to survive for several hours in 90% adult rat serum. With only a few exceptions, O7:K1 and O18:K1 strains were serum resistant and virulent, whereas O1:K1 strains were serum sensitive and avirulent. Serum sensitivity was due to the classical complement pathway. K1 strains of all three O serotypes were resistant to the alternative complement pathway. O7:K1 and O18:K1 cells were killed efficiently after the classical pathway was triggered by specific antilipopolysaccharide antibodies. However, killing of O1:K1 bacteria by the classical pathway system did not require antibodies. Isolated O1-lipopolysaccharide fixed complement more efficiently than did isolated O7- or O18-lipopolysaccharide, suggesting that the differences in the chemical structure of the O antigens are responsible for the observed differences in complement sensitivity. In combination with epidemiological data, the results indicate that antibody-independent classical pathway activation provides an important defense mechanism for newborns against certain gram-negative infections.

Antigens, Bacterial↗
PubMed1984

Two-dimensional gel pattern of proteins synthesized de novo in lymphoid organs of the mouse.

Mice were injected intravenously with 1 mCi of [35S]methionine, and serum and tissue were sampled 4 h later. Serum and lymphoid and non-lymphoid tissues had all incorporated enough labeled methionine to allow radiofluorographic detection on two-dimensional gels of proteins synthesized de novo and, by comparing radiofluorographs, we could distinguish spots peculiar to a given tissue from others more ubiquitous. We selected a protein of 17-kDa apparent molecular mass and pl about 4 to demonstrate tissue specificity. All patterns obtained for thymus samples yielded this spot; in all other immunologically relevant sites it was missing or weak. It also was not detected on gels previously obtained from lymphocyte subpopulations biosynthetically labeled in vitro. The labeling method described here will be especially helpful for characterizing cell populations that cannot be radiolabeled under cell-culture conditions. In contrast to detection methods that detect all proteins of a sample, in vivo labeling allows specific recognition of de novo synthesized proteins. Therefore it will facilitate comparison and detection of proteins produced by an animal in response to a given treatment.

Animals↗
PubMed1983

Induction of bacteremia in newborn rats by Escherichia coli K1 is correlated with only certain O (lipopolysaccharide) antigen types.

A total of 95 Escherichia coli strains (O1:K1, O7:K1, or O18:K1), obtained from different sources of human infections and from healthy individuals, were analyzed for the ability to cause bacteremia after colonizing the gut of newborn rats. Strains of all three serotypes were able to multiply extensively in the gut after oral inoculation and to translocate (in small numbers) to the mesenteric lymph nodes. With only few exceptions, O7:K1 and O18:K1 strains were able to cause bacteremia, while O1:K1 strains could not. Mixed-infection experiments revealed that the bacteria present in the blood during a case of bacteremia are in most cases the descendants of one cell that has multiplied extraintestinally after translocation to the mesenteric lymph nodes. It appears that virulent O7:K1 and O18:K1, but not avirulent O1:K1, bacteria are able to multiply directly in the bloodstream of the newborn rats. No correlation between virulence and the source of isolation of the different strains was observed. Disease isolates thus do not seem to differ from fecal isolates of the same serotype in special virulence properties. The differences in virulence among different O serotypes of K1 E. coli observed in the rat model were comparable to their relative frequency of isolation from meningitis in newborn children.

Animals↗
PubMed1983

Role of the capsule and the O antigen in resistance of O18:K1 Escherichia coli to complement-mediated killing.

Epidemiological data show that O18:K1 Escherichia coli is a common cause of neonatal bacteremia and meningitis. These bacteria were capable of multiplying in the bloodstream of newborn rats and were resistant to the bactericidal effects of complement in the absence of specific antibodies. The roles played by the O antigen and the K antigen in complement resistance were analyzed by comparing the bactericidal effects of normal sera and of sera deficient in various complement components or in immunoglobulins. These sera were tested on O18:K1 bacteria and on mutants lacking either the lipopolysaccharide O antigen or the K1 capsular polysaccharide. In addition, O1:K1 cells, which can cause pyelonephritis but which are rare in newborn meningitis and which do not multiply in the bloodstream of newborn rats, were also examined. Different mechanisms of protection against the alternative and classical pathways were recognized: K1-positive cells were resistant to the bactericidal activity of sera deficient in classical complement pathway components, whereas K1-negative cells were sensitive to these sera. Based on these results and on those from complement fixation assays, the K1 sialic acid polysaccharide impedes the activation of, and thus protects the bacteria against, the alternative complement pathway. Not only the K1-negative mutant cells but also O1:K1 bacteria and mutants lacking the O18 oligosaccharide repeating units of the lipopolysaccharide were sensitive to the classical complement pathway. These bactericidal effects were observed even in the absence of specific antibodies. It is proposed that both the K1 capsule and the O18 oligosaccharide restrict antibody-independent classical pathway activation by shielding deeper structures on the cell membrane that are capable of activating this pathway.

Animals↗
PubMed1981

Function of phospholipids in Escherichia coli. Influence of changes in polar head group composition on the lipid phase transition and characterization of a mutant containing only saturated phospholipid acyl chains.

The cls mutation conferring a defect in cardiolipin synthesis (Pluschke, G., Hirota, Y., and Overath, P. (1978) J. Biol. Chem. 253, 5048-5055) has been introduced into an Escherichia coli strain defective in unsaturated fatty acid synthesis in order to study the effect of changes in polar head group composition on the ordered in equilibrium fluid phase transition of the membrane phospholipids. The defect in cardiolipin formation is compensated by an increase in phosphatidylglycerol content, resulting in a decrease of the midpoint of the phase transition by 6 degrees C. Starvation of the cls mutant strain for the unsaturated fatty acid supplement leads to the incorporation of saturated acyl chains of reduced average length into the phospholipids, and growth is inhibited although the membrane remains in a fluid state. A revertant of this strain is described which retains the parental fabB-, fadE-, and cls markers and grows in the absence of an unsaturated fatty acid supplement. A cls+ derivative of the revertant can multiply in a restricted temperature range (35-43 degrees C). It contains only saturated phospholipid acyl chains of an anomalously short average length of 14 carbon atoms but has the same polar head group composition as wild type E. coli. The results demonstrate that, under defined conditions, saturated acyl chains of reduced length are functionally equivalent to unsaturated chains.

Cardiolipins↗
PubMed1981

Structure of Escherichia coli membranes. Glycerol auxotrophs as a tool for the analysis of the phospholipid head-group region by deuterium magentic resonance.

Glycerol selectively deuterated at various positions was synthesized and supplied to the growth medium of Escherichia coli strain T131 GP, which is defective in endogenous glycerol synthesis as well as glycerol degradation and lacks the ability to synthesize cardiolipin. The procedure enables the stereospecific labeling of the membrane phospholipids (approximately 80% phosphatidylethanolamine, approximately 20% phosphatdylglycerol). Deuterium magnetic resonance spectra were obtained for cell membranes and lipid dispersions either from total lipid extractions or from purified phosphitidylglycerol or -ethanolamine. When glycerol deuterated at various positions was used, all resonances of the phospholipid glycerol backbone and the terminal glycerol moiety in phosphatidylglycerol could be assigned. The results indicate that the molecular conformation of the glycerol backbone is independent of the phospholipid species investigated and is also not altered by the presence of high amounts of membrane proteins. For the quantitative interpretation of the deuterium magnetic resonance splittings, a model is proposed which assumes essentially free rotation around the glycerol C(2)-C(3) bond combined with an asymmetric and restricted jump process around the C(1)-C(2) bond. This model is compatible with known X-ray structures of phospholipids molecules. The two deuterons of both the glycerol backbone C(1) and C(3) segments were found to be magnetically inequivalent. Stereoselective monodeuteration eliminated one set of quadrupole splittings in both cases.

Cell Membrane↗