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P Gronski

Publications and source records attributed to P Gronski.

At least 19 recordsLinked to original sources
PubMed2006

IgG dimers in multidonor-derived immunoglobulins: aspects of generation and function.

Immunoglobulin G (IgG) concentrates for therapeutic purposes, like passive immunotherapy, supplementation in inherited or acquired deficiencies or immunomodulation, are prepared from multidonor-derived plasma pools. They usually contain varying amounts of dimeric IgG. The essential factor influencing dimer formation is the pool size; in addition, molecular properties of IgG and a variety of production process- and formulation-specific parameters are important. Numerous experimental findings suggest that dimers are predominantly generated by interactions of idiotypic and anti-idiotypic antibodies (Ids, anti-Ids). Ab-inherent crossreactivity, frequency distribution of both the affinities for particular Id-anti-Id interactions and the corresponding dimer concentrations still have to be elucidated. All these parameters influencing molecular features and functional activity of IgG dimers hamper the assay-dependent measurement of biological efficacy and correlation of total IgG content. A more detailed understanding may help to better control the dual nature of dimer-dependent biological activity comprising both undesirable (e.g., hypotension) and desirable effects of dimeric IgG (blockade of the reticuloendothelial system, RES, in immune thrombocytopenic purpura, ITP). These effects are detectable in in vitro and in vivo models and are thought to be of relevance for humans.

Animals↗
PubMed2003

Indications of neutralising anti-idiotypic antibodies and selective proteolytic fragmentation in polyclonal anti-D IgG preparations.

Proteolytic fragmentation is the only suggested cause of potency losses during storage of liquid human polyclonal anti-D Ig. Besides the effect of fragmentation, we have investigated the potential contribution of neutralising anti-idiotypic antibodies (anti-Ids). Potency changes during storage and/or upon pH reduction in anti-D IgG batches with or without addition of plasminogen and urokinase were quantitatively analysed by the autoanalyser (AA) method or by a special procedure of flow cytometry (FC). Moreover, simultaneous changes of the molecular size distribution pattern have been determined by size exclusion chromatography. In contrast to the AA procedure, the particular FC methodology was found to be almost insensitive to proteolysis comprising up to 30% of total IgG. Data interpretation was based on the assumption that both assays cannot detect Ids with neutralised paratopes. In the absence of detectable neutralisation (functional absence of anti-Ids), it could be demonstrated that the anti-D IgG subpopulation is more sensitive to fragmentation by endogenous protease as compared to the unrelated bulk. However, both methods detected batch- and assay-dependently variable potency losses during storage. Moreover, the increase of potency induced by pH reduction correlated with the increase of monomeric IgG, essentially on the expense of dimers. This finding was interpreted to indirectly indicate the neutralising action of anti-Ids known to be the major driving force of dimer formation in polyclonal IgG. A more or less pronounced pH-dependent potency increase was also detectable in three arbitrarily selected batches of two other manufacturers. The data allows to assume that anti-Id-mediated neutralisation can significantly contribute to losses of anti-D potency. In addition, it turned out that anti-D plasma itself can be the source of anti-Ids.

Antibodies, Anti-Idiotypic↗
PubMed1991

Comparative quantitation of anti-viral titers of polyclonal human IgG and (Fab')2 preparations by an enzyme-linked immunosorbent assay (ELISA): pitfalls of using appropriate second antibodies.

When comparing anti-viral antibody titers of intravenous immunoglobulins (i.v.-Igs) comprising two 7S IgG preparations and one corresponding (Fab')2 product, the specificity of the second ab-peroxidase conjugate employed in the ELISA was found to be decisive for the experimental results. Abs specific for the whole IgG molecule, in contrast to the (Fab')2 specific ones, obviously contain populations recognizing epitopes localized on C gamma 2 and C gamma 3 domains, and, therefore, cause an underestimation of titers of the (Fab')2 preparation.

Antibodies, Anti-Idiotypic↗
PubMed1990

Physiochemical features of monoclonal idiotype-anti-idiotype complexes: importance of inter-chain disulfides for size distribution patterns and molecular geometries.

Cyclic tetramers represent the preferentially formed complexes of a murine monoclonal idiotype-anti-idiotype (Id-anti-Id) system consisting of IgG antibodies or (Fab')2 fragments at micromolar concentrations. The cleavage of inter-chain disulfides of both Id and anti-Id caused the predominant generation of cyclic dimers at the expense of larger aggregates, suggesting with regard to already published data that the hinge located interheavy-chain disulfides are essential for the strain.

Antibodies, Anti-Idiotypic↗
PubMed1988

C4-binding protein prevents spontaneous cleavage of C3 in sera of patients with hereditary angioedema.

We have studied the effects of polyclonal monospecific Fab' preparations against C1r, C1s, C1INH, C4, C4bp, and fragment Bb of factor B on complement activation in NHS and HAES. Furthermore, we have investigated complement activation in these sera after addition of purified C1s and purified C4bp. Blocking C1INH induced a spontaneous activation of the classical pathway in NHS and to a lesser extent in HAES. Addition of p-C1s resulted in a strong C3 conversion in NHS, but not in HAES. However, after the blocking of C4bp in HAES, addition of p-C1s produced a total C3 consumption. The ration of the protein concentration of C4bp to hemolytically active C4 was eight times higher in HAES than in NHS. This increased ratio may account for the resistance of HAES to the C1s induced C3 cleavage in our in vitro system and the stability of C3 in HAE despite C4 and C2 consumption in vivo.

Angioedema↗
PubMed1988

E. coli derived human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) available for clinical trials.

Recombinant human GM-CSF has been expressed as a fusion protein in E. coli in the form of inclusion bodies. Using denaturing agents, acid cleavage and sulfitolysis, the biologically inactive GM-CSF protein could be highly purified and additionally renaturated under suitable reoxidizing conditions. The thorough repair of the two disulfide bridges could be confirmed by sequencing fragments obtained by tryptic digestion. Refolding of the molecule has been studied by CD spectrometry and identity by Western blotting and SDS-PAGE analysis. As could be demonstrated, full biological activity (colony-forming assay with fresh human bone marrow cells) was restored during renaturation of the GM-CSF protein. Further proof of biological equivalence of the E. coli-derived protein with a yeast-derived biologically active rh GM-CSF has been published elsewhere.

Circular Dichroism↗
PubMed1988

On the nature of IgG dimers. I. Dimers in human polyclonal IgG preparations: kinetic studies.

Human polyclonal IgG for therapeutic or prophylactic purposes is usually prepared from pooled plasmas taken from more than 1000 donors. After storage in solution under physiological conditions for a sufficiently long period, a considerable percentage of dimers (approx. 10-30% [w/w] at a protein concentration of approx. 160 mg/ml) represents the main component of aggregates in contrast to the essentially monomeric IgGs of monoclonal or single donor origin. Analysing the kinetics of monomer-dimer equilibration suggests assuming approx. 10(6) different antibody (ab) populations interacting independently and simultaneously with a specific partner of the reaction. Concerning the average apparent (functional) equilibrium constant of association, Kapp., a distinction could be made between two main populations characterized by values in the range if approx. 2.5-3.0 X 10(10) M-1 and 1.0 X 10(12) M-1, respectively. The results were obtained by computer simulation, taking an association rate constant, k+1, of 5 X 10(5) M-1 s-1 as a basis. Since the main part of the individual populations was found to interact Fc-independently via Fab-located binding sites, we suppose that the dimers are for the most part complexes of idiotypic (Ids) and anti-idiotypic abs (anti-Ids). Moreover, dimerization seems to be mainly a bivalent binding reaction, at least at the experimental concentrations. The results are in line with the concept of an idiotypic network regulation in man.

Animals↗
PubMed1988

On the nature of IgG dimers. II. Idiotype--anti-idiotype complexes of polyclonal and monoclonal origin: size distribution patterns and molecular geometries.

Electron micrographs of a fraction containing dimers isolated from pooled human polyclonal immunoglobulin G (IgG) suggest essentially a cyclic geometry compatible with bivalently associated monomers. It is obvious that such a structure can be produced by idiotype (Id)--anti-idiotype (anti-Id) interactions where the latter are able to neutralize certain combining site related Id functions. Accordingly, antibody (ab) activities against tetanus toxoid (tt) and rubella antigen (ag) were found to be almost exclusively confined to the monomeric molecules in preparations composed of monomers and dimers only. Moreover, electron micrographs of complexes prepared from a murine monoclonal Id as well as anti-Id reveal the presence of ring complexes, especially of cyclic tetramers. Gel filtration patterns of mixtures containing equimolar concentrations (concns) of such abs (1.6 x 10(-6) M) show, correspondingly for 9 different Id--anti-Id pairs and therefore probably representing a more common feature, mainly the formation of even-numbered complexes, especially tetramers. That is basically in accordance to an equilibrium model developed by Archer and Krakauer but not from a quantitative point of view because non-ideality terms had not been originally included. Despite taking strain energies determined by Schumaker et al. for cyclic complexes of polyclonal rabbit abs and a bivalent hapten into account for computation of size distribution patterns, the predominant formation of dimers was, nevertheless, again predicted by the modified theory in contrast to the experimental results. Fundamental conformity could only be achieved by further decreasing one of the statistical factors, namely the ring closing factor, which theoretically influences the generation of cyclic dimers. Therefore, referring to the experimental results of Schumaker et al., we postulate a strain energy well above 700 cal/mol for cyclic dimers produced by interacting Ids and anti-Ids. In general, the findings confirm predictions based on the interpretation of recent kinetic studies.

Animals↗
PubMed1987

Fc fragments of human IgG may influence allergic reactions.

The antiallergic effect of Fc fragments prepared from human polyclonal IgG was investigated in several experimental models. In an in vitro assay, isolated ileum of cynomolgus monkeys was sensitized with serum from atopic patients. In six of fifteen monkeys the subsequent addition of specific allergens reproducibly resulted in an ileum contraction measured in the Schultz-Dale apparatus. In all six positive monkeys pre-treatment of the isolated ileum with Fc (papain) before sensitization inhibited ileum contraction. Fc (plasmin) or Fc (pepsin) was less or not effective, aggregated IgG, monomeric IgG, sulfonated IgG, the Fc-free F(ab')2 moiety, or albumin had no significant or no reproducible inhibitory effect. In an in vivo assay passive cutaneous anaphylaxis was studied in 28 cynomolgus monkeys. Different dilutions in PBS of the particular specific allergens were subsequently administered to sensitized skin sites, simultaneously to i.v. injection of Evans blue. The degree of the local allergic (passive cutaneous anaphylactia) reaction was evaluated by measuring the diameter of the blue area at the dermal injection sites. About 50% of sera induced positive reaction in monkeys. Compared to the control (application of PBS), the degree of the positive reactions could be reduced by about 50% if Fc (papain) (optimal dose 25 mg/kg body weight) was injected i.v. either 2 h before or after local sensitization. No significant inhibitory effect could be found with the Fab moiety of IgG. Follow-up studies showed that the inhibitory effect of Fc lasted for about 10 days. The pharmacological basis of the observed antiallergic action of Fc is not yet understood.

Animals↗
PubMed1987

Quality criteria for i.v. immunoglobulins: importance of tests and product properties.

Quality criteria for i.v. immunoglobulins (i.v. Igs) are critically discussed laying emphasis on spontaneous anticomplementary activity (aca) and size-dependent composition. Considering the percentage of fractions obtained by gel filtration (Ultrogel AcA 34), however, the monomeric IgG containing fraction contributed the major part of aca under the experimental conditions chosen. Subclass IgG3 seems to contribute considerably to aca. No correlation was found between aca and the percentage of fractions containing components larger in size than IgG dimers in various commercially available Igs. Moreover, the subclass distribution in different batches of individual products showed considerable variations. The amount of IgG4 is correlated with that of IgA in chemically unmodified products relatively poor in IgA (approx. less than or equal to 10 mg/5 g Ig), indicating that attempts to reduce IgA consequently result in removal of IgG4.

Germany, West↗
PubMed1986

The functional inhibition of activated C1 inhibitor in normal human serum causes spontaneous consumption of the complement components C2, C3, C4, and factor B.

The human complement components C1r, C1s, C4, C3, factor B, and/or activated C1INH were functionally blocked in normal human serum (NHS) and EGTA- or EDTA-treated NHS by polyclonal monospecific Fab'-fragments to the individual components. The results of inhibition experiments are compatible with the formation of a classical pathway fluid-phase C3 convertase (C4b2a) spontaneously generated by the inhibition of activated C1INH. This process in both NHS and EGTA-NHS was accompanied by the consumption of C2, C4, C3, and factor B but only by poor enhancement of C5 conversion. Blocking subcomponent C1r, completely inhibited spontaneous activation of the complement components, indicating that the control of C1r hydrolysis is the essential role of activated C1INH as a regulator of C1 activation in NHS. Non-complement serum proteases were inactive during the initiation of the activation process. The presence of blood cells during functional inhibition of activated C1INH in NHS slightly decreased the consumption of C3 but not of C2 and C4.

Angioedema↗
PubMed1986

Quality criteria for i.v.-immunoglobulins: importance of tests and product properties.

Quality criteria for i.v.-immunoglobulins (i.v.- Igs) are critically discussed laying emphasis on spontaneous anticomplementary activity (aca) and size-dependent composition. Considering the percentage of fractions obtained by gel filtration (Ultrogel AcA 34), however, the monomeric IgG containing fraction contributed the major part of aca under the experimental conditions chosen. Subclass IgG3 seems to contribute considerably to aca. No correlation was found between aca and the percentage of fractions containing components larger in size than IgG dimers in various commercially available Igs. Moreover, the subclass distribution in different batches of individual products showed considerable variations. The amount of IgG4 is correlated with that of IgA in chemically unmodified products relatively poor in IgA (approx. less than or or equal to 10 mg/5 g Ig), indicating that attempts to reduce IgA consequently result in removal of IgG4.

Complement C3↗
PubMed1985

The modulation of immune complex aggregation by classical pathway-mediated reactions.

Classical pathway (CP)-triggered reactions of complement-modulated immune complex (IC) aggregation (tetanus toxoid/human anti-tetanus toxoid-IgG; ICs of equivalence) were investigated turbidimetrically during the early stages of reaction. Monospecific Fab'- or Fab-fragments (rabbit) directed against certain complement components were used to block the complement function in normal human serum (NHS). Additionally, parts of the reactions were studied using purified complement components. C1q in serum generated by the addition of EDTA as well as purified C1q were found to increase the IC aggregation. In contrast to C1q, macromolecular C1 is able to inhibit IC aggregation, whereas additional participation of C-1 INH reversed this process. The cooperation of the remaining CP proteins (C4, C2, C4bp, and I) reconstituted the inhibition capacity of the complement. Whereas C3 supported significantly inhibition, a significant influence of other effector pathway (EP) components (C5-C9) was not detectable turbidimetrically.

Antigen-Antibody Complex↗
PubMed1984

The influence of classical pathway components during alternative pathway--modulated immune complex aggregation: the role of C1 INH.

Alternative pathway (AP)-triggered reactions as well as classical pathway (CP)-mediated ones, were investigated turbidimetrically and/or immune electrophoretically, either in the presence or in the absence of in situ-generated immune complexes (ICs; tetanus toxoid/human anti-tetanus toxoid-IgG; ICs of equivalence) during the early stages of reaction. Monospecific Fab'- or Fab-fragments (rabbit) were used to block the complement function in normal human serum (NHS). C1q, functionally available following the addition of ethylene-glycol-bis-(beta-aminoethyl ether), N,N'-tetraacetic acid to NHS (EGTA-NHS), was found to increase the IC aggregation, thereby producing a biological surface upon which AP-dependent proteins were deposited. The functional inhibition of C1INH caused a C1s-mediated C3 conversion irrespective of the fact whether C1s was incorporated within macromolecular C1 (NHS) or dissociated from it (EGTA-NHS), thus, in the latter case inhibiting the AP-dependent portion of turbidity. It seemed probable that C3 conversion was effected by a fluid-phase CP C3 convertase. This process, normally counteracted by C1INH, worked more efficiently in EGTA-NHS than in NHS, indicating that the C1s-mediated reactions, initiated by presently unknown mechanisms, were less extensively regulated outside of the Ca2+-dependent C1 complex. The study demonstrates that in EGTA-NHS, too, where AP-triggered reactions have usually been investigated, sections of CP activation may play an important role, especially in situations where the function of C1INH is restricted.

Antigen-Antibody Complex↗
PubMed1984

Immunotoxins--theoretical and practical aspects.

The clinical application of immunotoxins is still hampered by a number of unsolved problems. These include difficulties associated with each of the components of the conjugates, i.e., antibody, coupling agent, and cytotoxic drug. Additional problems are caused by the conjugate formation itself. Major obstacles are the lack of data relating to the pharmacokinetics of immunotoxins and their fate after binding to the target cells. This article reviews these problems and indicates possible solutions, including two suggestions for model immunotoxins.

Antibodies, Monoclonal↗
PubMed1984

Basic relationships in precipitating antigen-antibody systems: a comparison of a simple theory with experiment.

Theoretical predictions from a simple theory for a homogeneous system with respect to some fundamental functional relations between amount of precipitate or extent of turbidity, initial antigen (Ag) respectively antibody (Ab) concentration, and solubility of immune complexes (ICs) are discussed in comparison with experimental results in a heterogeneous system. Experiments were performed with the aim to render possible and intuitive picture of the relationships considering different aspects. It was found that the theory derived from the equilibrium state of the reaction is also able to describe the non-equilibrium state in a qualitatively correct manner. The influence of Ab affinity on precipitation is discussed. Moreover, it is shown that the theory developed by Pauling et al. in the 1940's although too simple in several details, does, however, consider the most important basic principles of more extensive theories developed by other authors. Precisely because of its simplicity, this model may be a useful help in the discussion of precipitate formation on a molecular level.

Antigen-Antibody Complex↗