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Biomedical subjects

T E Michaelsen

Publications and source records attributed to T E Michaelsen.

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

The extended hinge region of IgG3 is not required for high phagocytic capacity mediated by Fc gamma receptors, but the heavy chains must be disulfide bonded.

Fc gamma receptor (Fc gamma R) phagocytosis and respiratory burst were induced by chimeric mouse-human anti-(4-hydroxy-5-iodo-3-nitrophenyl) acetyl IgG3 antibodies with mutations in hinge and/or in CH1 region. IgG3 mutants with different hinge length ranging from 47 to 0 amino acids, an IgG3 molecule with an artificial hinge of just one cysteine residue (HM-1), and two hybrid IgG3 molecules with IgG4 hinge or IgG4 CH1-hinge were tested. Using the monocytic cell line U937 as effector cells, the mutated IgG3 molecules were very similar, revealing high activity, while the IgG3/IgG4 hybrids revealed a slightly reduced activity. However, the hingeless (0-h) mutant was negative, except after interferon-gamma stimulation when it became slightly positive. Interestingly, HM-1 was as active as the IgG3 mutants. With polymorphonuclear leucocytes (PMN) as effector cells we obtained some day-to-day variations, but all the IgG3 mutants were highly active, with the two shortest hinge mutants somewhat less active. The IgG3/IgG4 hybrid molecules revealed an intermediate activity, while IgG4 wild-type and the 0-h mutant were negative. However, the HM-1 molecule revealed an activity similar to that of the IgG3 mutants. The phagocytic activity of U937 was inhibited by monomeric IgG, indicating the importance of Fc gamma RI. In contrast, with PMN both blockage of Fc gamma RII and cleavage of Fc gamma RIII were required to significantly reduce the phagocytosis and respiratory burst, thus showing that both receptors contribute to the effect. These results demonstrate that the extended IgG3 hinge region is not necessary for a high phagocytic activity and that the major structural importance of the hinge is to connect the two heavy chains in this region.

Disulfides↗
PubMed1993

Human IgG3 can adopt the disulfide bond pattern characteristic for IgG1 without resembling it in complement mediated cell lysis.

In this paper we describe the construction of mouse-human IgG3 mutant antibodies resembling IgG1 in their disulfide bond pattern between the heavy and light chain (H-L) and between the two heavy chains (H-H). The effector functions of these mutant antibodies were compared to normal IgG3 and IgG1. Changing only the disulfide bond pattern between the heavy and light chains did not alter the ability to induce complement mediated cell lysis (CML), regardless of the amount of corresponding antigen that had been introduced to the surface of the target cells. However, alteration of the disulfide bond pattern between the two heavy chains had a large effect on CML due to shortening of the hinge from 62 to 15 amino acids. No difference between the mutants and normal antibodies in antibody-dependent cell-mediated cytotoxicity (ADCC) was observed. This suggests that IgG3 can adopt the H-L disulfide bond pattern of IgG1 without obtaining the CML activity characteristic for IgG1.

Amino Acid Sequence↗
PubMed1993

Human IgG3 is decreased and IgG1, IgG2 and IgG4 are unchanged in molecular size by mild reduction and reoxidation without any major change in effector functions.

Purified proteins of the four human IgG subclasses were reduced under neutral conditions to break the interchain S-S bonds, followed by dialysis to allow reformation of S-S bonds (pr/o treatment). The IgG1, IgG2 and IgG4 proteins apparently reformed native molecules by pr/o treatment, while IgG3 formed molecules with significantly smaller size, as measured by HPLC gel filtration, compared to the autologous native proteins. The degree of shrinking of the pr/o IgG3 molecules varied and was most pronounced at low protein concn. In addition, the temp and the concn of reducing agent during the pr/o treatment had some influence on the molecular size. The effect is probably due to a conformational change of the 62 amino acid long hinge of IgG3. The effector activity of pr/o IgG2 and pr/o IgG3 was studied by employing chimeric, mouse V and human C regions, monoclonal antibodies with the same NIP-binding properties. Thus, the interaction between IgG and the complement system was unchanged both for pr/o IgG2 and pr/o IgG3, while the Fc-receptor-mediated antibody-dependent cellular cytotoxicity (ADCC) was depressed to the same degree for both pr/o IgG2 and pr/o IgG3. Conclusively, the alteration of the conformation of the IgG3 molecule by pr/o treatment had no major influence on its effector functions.

Animals↗
PubMed1993

A low serum concentration of mannan-binding protein is not associated with serogroup B or C meningococcal disease.

The mammalian C-type serum lectin, mannan-binding protein (MBP), may induce C1q- and antibody-independent activation of the classical pathway of complement. Accordingly, MBP is considered as a member of the complement system. Complement deficiencies have been found with increased frequency in patients with meningococcal disease. Therefore, we investigated the MBP levels in patients with meningococcal disease. Ninety-nine Norwegian individuals (age 12-21 years) who survived severe systemic disease caused by serogroup B or C meningococci were investigated. No significant differences were observed in the MBP concentration between patients with serogroup B (n = 76) or C (n = 25) disease and healthy blood donor controls (n = 40) (P > 0.05). The frequency of patients with low levels of MBP (< 100 micrograms/l) was 10.1%. This was not different from controls (12.5%). Thus, low MBP concentrations do not appear to predispose to serogroup B or C meningococcal disease.

Adolescent↗
PubMed1993

Detection of pathogenic Yersinia enterocolitica in foods and water by immunomagnetic separation, nested polymerase chain reactions, and colorimetric detection of amplified DNA.

A two-step polymerase chain reaction (PCR) procedure with two nested pairs of primers specific for the yadA gene of Yersinia enterocolitica was developed. The PCR assay identified all common pathogenic serogroups (O:3, O:5,27, O:8, O:9, O:13, and O:21) from three continents and differentiated pathogenic Y. enterocolitica from Y. pseudotuberculosis and from a variety of nonpathogenic yersiniae representing 25 serogroups and four species. The performance of the method was evaluated with seeded food and water samples. We compared two procedures for sample preparation prior to PCR: one was based on immunomagnetic separation of the target bacteria from the sample, using magnetic particles coated with immunoglobulin antibodies to Y. enterocolitica serogroup O:3, and the other method consisted of a series of centrifugation steps combined with proteinase treatment. Regardless of the method used, the PCR assay was capable of detecting 10 to 30 CFU/g of meat in 10(6)-fold excess of indigenous bacteria. When the samples were enriched overnight in a nonselective medium, the sensitivity was increased to approximately 2 CFU/g, except for samples with an extremely high background flora (> 10(7) CFU/g). We compared gel electrophoretic detection of PCR products with a colorimetric detection method designated DIANA (detection of immobilized amplified nucleic acids), which enabled easy visualization of amplified fragments in a microtiter plate format with an optical density reader. DIANA and gel electrophoresis showed complete concordance in their discrimination between positive and negative samples. The combination of immunomagnetic separation, nested PCR, and DIANA makes possible the development of a fully automated analytic process which requires a minimum of laboratory manipulations.

Adhesins, Bacterial↗
PubMed1992

Unexpected interaction of some anti-TNP hybridoma antibodies with Superose HPLC gel filtration resins.

Five different hybridoma antibodies (the isotypes IgG1, IgG2a and IgG2b), reactive with TNP, showed increased elution times when gel filtered on a Superose-12 HPLC column corresponding to apparent molecular weights ranging from 54 kDa to 120 kDa compared to the normal of 150 kDa. One of the antibodies (C1901-B4) was studied in detail showing that the unusual gel filtration behaviour was localized to the Fab part of the molecule. SDS-PAGE analysis showed that the intact antibodies had normal molecular weights. Gel filtration on a Toyosoda G-3000SW HPLC column and Sephadex G-150 as well as Sepharose 6B generally showed normal elution times. These results support the hypothesis that the retardation on the Superose gel is probably due to aromatic interactions between amino acid residues supposedly exposed in the hypervariable region (i.e., the antibody combining site) of the antibody, and the gel matrix which is rich in ether O-atoms created during the manufacturing process. If this hypothesis is correct one might expect such interactions between Superose resins and antibodies of many different specificities in which aromatic amino acids are exposed in the combining sites.

Chromatography, Gel↗
PubMed1992

The binding parameters of radiolabelled monoclonal F (ab')2 and Fab' fragments relative to immunoglobulin G in reactions with surface-bound antigens.

The binding parameters of iodine-125-labelled intact monoclonal immunoglobulin G (IgG), F(ab')2 and Fab' fragments were compared. The study was carried out with the two monoclonal antibodies (MoAbs) K13 and K16 specific for human Ig light chains kappa and lambda, respectively. When testing the 125I-MoAbs against monodisperse polymer particles coated with the specific antigens, the Ka for the F(ab')2 fragments were similar to that for IgG, while the Ka for the Fab' fragments were reduced to 10%-20% of that for IgG. The number N of effective target sites revealed with Fab' was higher than with F(ab')2 and IgG, presumably because less surface area is occupied by the small Fab' molecules. The immunoreactive fraction F ranged according to IgG greater than F(ab')2 greater than Fab'. The explanation of the moderate difference between the Ka of the monoclonal Fab' and the divalent IgG and F(ab')2 was that the divalent molecules were not divalently attached to the particles. When testing the same antibody preparations against human lymphoma cells producing Ig with light chains kappa or lambda, the binding results were less reliable than when particles were utilised, presumably due to antigen shedding. Different MoAbs vary in their loss of immunoreactivity due to enzymatic degradation and the radiolabelling procedure. The preparation of the radiolabelled fragments should therefore be optimized for each MoAb, and evaluation is necessary before injection. Artificial targets with a low leakage of antigen, like the monodisperse polymer particles here applied, are recommended for the in vitro evaluation of the immunoreactivity of labelled MoAb preparations.

Animals↗
PubMed1992

Antibody dependent cell-mediated cytotoxicity induced by chimeric mouse-human IgG subclasses and IgG3 antibodies with altered hinge region.

A matched set of chimeric mouse-human NP-antibodies were studied for the capacity to induce cell mediated cytotoxicity (ADCC) by normal peripheral blood NK/K cells. The target cells were sheep red blood cells (SRBC) sensitized with the haptens NP or NIP. All four IgG subclasses and several IgG3 variants with altered hinge were tested for ADCC activity. The hierarchy of the ADCC capacity among the subclasses was found to be IgG3 greater than IgG1 greater than IgG4 greater than IgG2. The superiority of IgG3 was only revealed at low effector cell:target cell ratio. The ADCC activity was for the most part unaltered by shortening the hinge region of IgG3 from 62 to 15 amino acids. Also, when the hinge region of IgG3 was mutated to become identical to that of IgG4, the ADCC activity was mainly unchanged. However, an IgG3 variant with deletion of all four hinge exons showed a depressed ADCC activity compared to the wild type. The IgG subclass pattern of complement-mediated lysis (CML) and ADCC is different and the capacity to induce CML and ADCC is changed differently by hinge region modification. Thus CML and ADCC have different structural requirements in the Fc region of IgG.

Amino Acid Sequence↗
PubMed1992

Antigenic change in a human IgG4-specific CH3 epitope upon binding of a monoclonal antibody against a neighboring IgG4-specific epitope.

Two sets of monoclonal antibodies (mAbs) probably reacting with two different epitopes in the CH3 domain of the human IgG4 molecule were studied. We observed that the commercially available mAb HP 6011 inhibited the antigen binding of the three mutually inhibitable mAbs, 40-A2, 41-E8 and 43-F11 (40-series), made by us. However, the 40-series mAbs, including those with similar affinity such as mAb HP6011, were not able to inhibit mAb HP 6011. When the 40-series mAbs were preincubated with IgG4, the mAb HP 6011 could partially displace these antibodies. This one-way inhibition indicates that upon binding mAb HP 6011 changes the antigenic structure of the IgG4 molecule by disrupting the epitope for the 40-series mAbs. A steric hindrance of this epitope by mAb HP 6011 is more unlikely, since the small Fab fragment of mAb HP 6011 also inhibited the reaction of the 40-series mAbs.

Antibodies, Monoclonal↗
PubMed1992

SCID-Hu mice immunized with a pneumococcal vaccine produce specific human antibodies and show increased resistance to infection.

Seventy-eight severe combined immunodeficiency (SCID) mice were administered intraperitoneally 1 x 10(7) to 9 x 10(7) human peripheral blood mononuclear cells (PBL) in five experiments. Human immunoglobulin G (IgG) was detected in 70 to 88% of these SCID-PBL-Hu mice after cell transplantation, and all four subclasses were present. The total concentration of human IgG varied from less than 1 to 10.2 g/liter. The SCID-PBL-Hu mice with high concentrations of human IgG regularly had mono- or oligoclonal human IgG bands in serum, as demonstrated by agarose gel electrophoresis. Of the SCID-PBL-Hu mice that were immunized with a 23-valent pneumococcal polysaccharide vaccine, 63 to 78% developed a significant human IgG antipneumococcal antibody response, whereas only very low levels of human IgM and no human IgA antipneumococcal antibodies could be detected. Twelve to twenty-two percent of the SCID-PBL-Hu mice showed signs of leakiness; these mice developed a significant mouse IgM antipneumococcal antibody response and no human antibodies. SCID-PBL-Hu mice were challenged intraperitoneally with 10 50% lethal doses of Streptococcus pneumoniae serotype 4 to study the protective effect of immunization with pneumococcal vaccine. The immunized SCID-PBL-Hu mice showed less bacteremia than did all control groups, and survival was 45 to 60%. None of the unimmunized SCID-PBL-Hu mice survived.

Adult↗
PubMed1991

The use of a hapten-Fab conjugate to sensitize target cells for antibody-dependent complement-mediated lysis and antibody-dependent cell-mediated cytotoxicity.

NIP-conjugated Fab' fragments from a rabbit hyperimmunized with sheep red blood cells (SRBC) were used to link the hapten NIP to target cells for antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent complement-mediated lysis (ADCML). Target cells (SRBC) labelled with this NIP-Fab' complex were compared with SRBC directly haptenized with NIP in ADCC and ADCML assays using a NIP specific IgG1 chimeric antibody. Both methods yielded almost identical results. Using the NIP-FAb' conjugate identical target cell haptenization was readily achieved from experiment to experiment. Using conjugates of different NIP/Fab' ratios it should be possible to study how such changes influence antibody effector functions.

Animals↗
PubMed1991

Human IgG subclass pattern of inducing complement-mediated cytolysis depends on antigen concentration and to a lesser extent on epitope patchiness, antibody affinity and complement concentration.

The relative complement-mediated lytic capability of the IgG subclass isotypes was studied using a matched set of mouse-human chimeric anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies. The subclass pattern was shown to be highly dependent on variations in antigen concentration and to lesser extent on variation in epitope patchiness, antibody binding affinity and complement concentration. In general, the IgG3 subclass was most effective in inducing cytolysis at the different conditions used and only at high antigen concentration did the IgG1 subclass mediated more efficient cytolysis than IgG3. The IgG2 isotype required a relative high antigen concentration to be cytolytic while the IgG4 isotype was not cytolytic at any of the conditions tested. These individual characters of each of the IgG subclasses makes it conceivable that a subtle system of immunoregulation exists among the subclasses.

Antibody Affinity↗
PubMed1991

Chimeric mouse human IgG3 antibodies with an IgG4-like hinge region induce complement-mediated lysis more efficiently than IgG3 with normal hinge.

We have altered the amino acid sequence of the hinge and the first constant domain (CH1) of mouse/human chimeric IgG3 antibodies by site-directed mutagenesis, so as to make the sequences identical to those of IgG4. All the mutant antibodies with altered hinge region were more active in complement activation and complement-mediated lysis than native IgG3. The mutations in CH1, however, did not alter the activity. This demonstrates the importance of the hinge region in modulating this effector function. The results show that the primary structure of neither CH1 nor the hinge of IgG4 is responsible for the lack of complement activation shown by this subclass.

Amino Acid Sequence↗
PubMed1990

C3, C4, and the terminal complement complex differ from C1q by binding predominantly to the antigenic part of solid phase immune complexes.

The binding of the C components C1q, C4, C3, the terminal C5b-9 complement complex (TCC) and S protein to immune complexes was studied. The hapten 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) conjugated to BSA was adsorbed to polystyrene plates and reacted with a human IgG3-mouse chimeric anti-NIP antibody. After addition of serum a dose-dependent binding of C1q, C4, C3, and TCC to the immune complexes was found. An increase in the amount of NIP-BSA was associated with an increase in the binding of TCC and a decrease in the binding of S-protein. After addition of soluble NIP only 4 to 6% of the anti-NIP antibody remained bound to the Ag. C1q showed diminished binding after addition of NIP, whereas C4, C3, and TCC quantitatively remained bound to the Ag. Binding of TCC to the immune complexes was also found in an alternative assay, in which the anti-NIP antibody was adsorbed to the solid phase before NIP-BSA and an additional layer of anti-NIP antibody were added. The supernatants from the solid phase assay were tested for C3 activation and formation of the fluid phase TCC (SC5b-9). Activation of the C3 was reflected in the fluid phase by a dose-dependent increase in C3 activation products. This was not seen for TCC despite increased binding to the solid phase.

Animals↗
PubMed1990

IgG subclass antibody responses to pneumococcal polysaccharide vaccine in splenectomized, otherwise normal, individuals.

Subclasses of IgG antibodies to pneumococcal polysaccharide serotype antigens 4, 6A, and 23F were measured before and 4 weeks after vaccination with pneumococcal vaccine in young individuals splenectomized because of trauma and in a control group. An ELISA technique was applied. IgG2 anti-pneumococcal antibodies predominated before vaccination, especially against serotypes 4 and 6A. The youngest individuals in the splenectomy group tended to have lower IgG2 anti-pneumococcal antibody levels than the older ones. Vaccination induced antibodies of the IgG1 and IgG2 subclasses, and in some individuals also of the IgG4 subclass. Splenectomy does not seem to influence the IgG subclass pattern of antipneumococcal antibodies.

Adolescent↗