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Biomedical subjects

T Hofstaetter

Publications and source records attributed to T Hofstaetter.

At least 19 recordsLinked to original sources
PubMed1985

Influence of different immunoglobulin G preparations on phagocytosis of Pseudomonas aeruginosa by polymorphonuclear granulocytes.

Commercially available immunoglobulin products suitable for intravenous application in humans are made by enzymatic or chemical modification of the IgG molecule. In order to examine, to which extent in vitro biologic functions of the IgG molecule are preserved in such preparations, specific antipseudomonal IgG from the rabbit was modified according to some of these procedures. The opsonizing activity of the different IgG preparations was evaluated in an in vitro system measuring the phagocytosis of Pseudomonas aeruginosa by rabbit granulocytes. The results demonstrated that the product Fab/Fc, which is made by papain or plasmin degradation of the IgG molecule, was still able to enhance phagocytosis but not to activate complement. The smaller papain- or plasmin-derived fragments Fab and Fc had no opsonizing activity. The pepsin-derived product F(ab')2 which possesses a divalent antigen binding site but lacks the Fc part, only enhanced phagocytosis when complement concentrations of more than 20% were present in the phagocytic system. Since the F(ab')2 fragment is not able to interact with Fc receptors or to activate complement via the classical pathway, the phagocytosis-enhancing activity of this molecule must be attributed to alternative complement pathway activation. In contrast to the enzymatically derived IgG preparations, a newly developed S-sulphonated IgG product was as efficient as unmodified IgG both in Fc- and complement-mediated opsonization.

Animals↗
PubMed1985

In vitro and in vivo activities of different immunoglobulin G preparations from the rabbit against Enterobacteriaceae.

Specific rabbit IgG, prepared against three enterobacterial strains, was modified according to procedures used for the production of human IgG preparations suitable for intravenous infusion. The resulting products were examined for their in vitro opsonic and in vivo protective activity against the respective bacterial strains. A sulfonated IgG preparation (S-sIgG) and the enzymatically derived fragments F(ab')2 and Fab/Fc were opsonic in vitro and provided in vivo protection against lethal enterobacterial infection in mice. Protection by Fab fragments varied in the different in vivo models.

Animals↗
PubMed1984

Evidence against a role for neutrophils in the clearance of small immune complexes.

The activation of human neutrophils (PMN) by immune complexes (IC) consisting of tetanus toxoid and the corresponding human antibodies was studied by varying IC properties like size, antigen: antibody ratio, Fc density, etc. While Ag: ab ratio seemed to have some importance, especially when complement activation was allowed, size appeared to be the decisive factor. Surprisingly, good stimulation of PMN was obtained only with very large IC and no plateau was reached with increasing size. PMN activation even by these very large complexes was inhibited in the presence of physiological IgG concentrations, but only if complement was omitted. Generation of IC in the presence of serum, on the other hand, prevented the formation of IC sufficiently large for PMN activation. Finally, in an IC clearance experiment in mice using 125I-labelled human serum albumin and mouse antibody, no radioactivity was found associated with blood PMN in spite of a rapid clearance. It is concluded that PMN are probably not involved in the physiological clearance of IC if these are considerably smaller than opsonized microorganisms.

Animals↗
PubMed1984

The interaction of IgG with Fc gamma receptors on human neutrophils.

The interaction of IgG with Fc gamma receptors on human PMN was studied by investigating the capacity of IgG and some of its fragments to mediate and inhibit, respectively, binding of immune complexes to these receptors and the subsequent activation of the cells. Fragments included Fab/Fc which is monovalent but contains the complete Fc region, Facb (divalent, C gamma 3 domains removed), Fc, pFc' (a C gamma 3 dimer) and a peptide corresponding to the greater part of one C gamma 2 domain (amino acids 278 through 333) including the carbohydrate side chains. F(ab')2 and Fab served as controls. The results indicate that human IgG interacts with human PMN Fc receptors predominantly through its C gamma 2 domains, while binding of rabbit IgG involves additional sites in C gamma 3. The C gamma 2 binding site on human IgG appears to be located between those for C1 and protein A and to contain lysine residues. For its correct exposure the C gamma 2 domains must be kept in the native conformation, in which both the hinge disulfide bridges and the carbohydrate moieties are involved. A role of the sugars in the actual binding, on the other hand, can be excluded.

Antigen-Antibody Complex↗
PubMed1984

Phagocytosis of immune complexes by human neutrophils and monocytes: relative importance of Fc and C3b receptors.

Human neutrophils and peripheral blood monocytes were studied for their Fc- and C3b-receptors after incubation of the cells with two types of immune complexes (IgG or F(ab')2. Immune complexes can bind to and activate phagocytes via Fc- and C3b-receptors. On mononuclear phagocytes an additional receptor is found that is able to interact directly with F(ab')2-complexes. C3b- and Fc-receptors can mediate ingestion of appropriately opsonized particles, C3b-receptors being at least as efficient as Fc-receptors. The extents of cell activation and phagocytosis in vitro depend on a number of variables, like complex size, ligand (antibody, C3b) density on the surface of opsonized particles, receptor number etc., which are not controlled in most studies and thus may explain the discrepant results reported in the literature. With the in vitro results obtained here, we cannot fully explain in vivo data obtained by various groups, where F(ab')2 antibodies yielded full protection against different types of infections. Therefore, great caution must be applied in all attempts to extrapolate in vitro data to the actual situation in vivo.

Animals↗
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↗
PubMed1983

The biological properties of immunoglobulin G and its split products [F(ab')2 and Fab].

Antibodies of the IgG class possess antibacterial, antiviral and toxin neutralizing properties and for this reason are administered prophylactically and therapeutically. In the case of the immunoglobulin preparations commercially available for i.v. application a basic distinction must be made between unsplit immunoglobulins and those antibody preparations obtained by enzymatic digestion, such as F(ab')2 or Fab antibodies. This survey deals with the largely experimental evidence describing the biological properties of these preparations. Administration of antibodies in the presence of the corresponding antigens leads to the formation of immune complexes in the organism. These immune complexes can activate, either directly or indirectly, the cellular and humoral systems which are involved in phagocytosis and the elimination of antigens, in the regulation of the body's own antibody production and in inflammatory reactions. As a result of their inability to interact with Fc receptors, immune complexes with F(ab')2 or F(ab) antibodies appear to be less active in the release of inflammation mediators from leucocytes and thrombocytes than immune complexes with unsplit immunoglobulins. These, on the other hand, can antigen-specifically and non-antigen-specifically suppress the immune system which is not the case for immune complexes with F(ab')2 or Fab antibodies. There are indications that these split products also occur in vivo due to the action of tissue and leucocyte proteases. Unlike Fab preparations, F(ab')2 antibodies have antibacterial and antiviral potencies similar to unsplit immunoglobulins, which is probably due to the ability of F(ab')2 molecules to activate complement, not by the classical but by the alternative pathway. Like Fab preparations, F(ab')2 molecules appear to be superior to unsplit IgG in the elimination of haptens. On account of the relatively long period of time unsplit immunoglobulins remain in the blood, they are well suited for prophylactic treatment and substitution over longer periods. The extent to which indications, obtained predominantly from experimental studies, of a reduced release of inflammation mediators, a lack of immune suppression and a lack of augmentation of IgG catabolism would advocate the use of F(ab')2 split products, especially for therapeutic purposes, can only be ascertained after prospective and comparative studies have been carried out.

Antigen-Antibody Complex↗
PubMed1983

Metabolism and non-random occurrence of nonnascent short chains in the DNA of Ehrlich ascites cells.

The DNA of Ehrlich ascites cells was labeled with radioactive thymidine using different labeling schedules: Incubation periods between 15 s and 4 h; pulse/pulse-chase experiments with pulses in the range of a few minutes; longtime incubation followed by a longtime chase (both in the range of 1 cell generation). From the purified DNA of the labeled cells a fraction (0.3-0.4%) of short chains was separated and partially fractionated by means of a hydroxyapatite thermochromatography procedure. The evaluation of the labelling patterns of the short chains indicated that less than 5% of them can be regarded as replication intermediates ('Okazaki pieces'). The rest, termed nonnascent pieces, exhibited a slow turnover. The life span of the nonnascent pieces was estimated to be about 1 cell generation. On helical DNA, nonnascent pieces were distributed in a non-random manner. Their preferential localisation was nearby sites which caused binding of the DNA, after purification, to nitrocellulose and which occurred about every 60-80 microns on the nuclear DNA of the cells.

Animals↗
PubMed1983

S-sulfonation: a reversible chemical modification of human immunoglobulins permitting intravenous application. I. Physicochemical and binding properties of S-sulfonated and reconstituted IgG.

S-sulfonation represents a reversible chemical modification of disulfide bonds by which under the special conditions chosen only about 2.2 cystine units per IgG molecule are cleaved. Physicochemical and functional evidence for reconstitution is presented. Molecules reconstituted in vitro or in vivo regain, within a few hours, a reactivity (antigen binding, immunoprecipitation, Clq-mediated cross-linking of immune complexes) comparable to equimolar control preparations.

Animals↗
PubMed1983

S-sulfonation: a reversible chemical modification of human immunoglobulins permitting intravenous application. II. Effect on Fc-mediated effector functions.

The generation and release of mediators of inflammation and anaphylaxis via activation of complement or of Fc receptor-bearing cells is held responsible for adverse reactions observed upon intravenous administration of standard immunoglobulins. They are caused by immunoglobulin G (IgG) effector functions predominantly located in the Fc region of the molecule and 'activated' by aggregate formation. Their functional activity depends on the correct conformation of the C gamma 2 domains of Fc and are therefore impaired or even abrogated by S-sulfonation of the hinge disulfide bonds, as demonstrated in this communication: S-sulfonated IgG (S-IgG) has no anticomplementary activity and does not interact with Fc-receptors anymore. After antigen binding, i.e. immune complex (IC) formation, sulfonated IgG is about half as potent as standard IgG in complement activation or phagocyte stimulation (human monocytes and granulocytes). The two activities synergize, however, so that in the presence of complement S-IgG IC are as effective phagocyte activators as standard IC. Moreover, S-sulfonation being the only chemical modification of immunoglobulins that is reversible, it can be demonstrated that all IgG effector functions important for antigen removal are regained by reconstitution of the disulfide bonds.

Antigen-Antibody Complex↗
PubMed1981

Mouse spleen cells are sensitized sheep red blood cells: an Fc-rosette-forming system allowing the detection of "activated" Fc structures.

From a variety of Fc receptor-bearing cell/sensitized red blood cell combinations, mouse spleen cells, and sensitized SRBC were selected as an Fc-specific EA rosette assay system because only this mixture combined a high percentage (about 50%) of rosette-forming cells with complete absence of spontaneous rosettes and showed no influence of complement on the rosette formation. From studies on the minimal structural requirement of IgG both for mediation and inhibition of EA rosettes using IgG and several well-defined fragments, it appeared that both the CH2 and the CH3 domain of Fc are needed for optimal interaction with the lymphocyte Fc receptor. Finally, it was demonstrated that the assay system is able to detect "activated" Fc structures (here: heat-aggregated IgG) and to differentiate between varying amounts of such structures.

Animals↗
PubMed1981

A microassay for nitroblue tetrazolium reduction by human neutrophils.

By adapting the nitroblue tetrazolium reduction test (NBT test) of neutrophil function to microtiter plates with correspondingly small incubation and dye extraction volumes, it has been possible to develop a microassay with the following advantages: 1. The cell number required for single standard assay has been reduced five- to tenfold to 1 X 105 PMN. 2. If little blood or few cells are available, this number can be further reduced to 1-5 X 104. 3. The test is rapid (less than 6 hours overall) and highly reproducible. 4. A single technician can process at least six plates = 576 single assays at a time. 5. Due to the small incubation volume, test substances can be saved. NBT reduction in this test is a function of both cell number and concentration of the stimulus (here: zymosan). Addition of serum, Mg+/-+/-, or EGTA/Mg enhances the response to zymosan while Ca+/- +/- at greater than or equal to 2 mM is inhibitory.

Dose-Response Relationship, Drug↗
PubMed1981

Pharmacokinetics of human interferon-beta in monkeys.

Human fibroblast-derived interferon (HuIFN-beta) was administered to cynomolgus monkeys i.m. (2 x 10(5) iu/kg), i.v. (2 x 10(5) iu/kg) or intrathecally (3 x 10(5) iu/monkey) or given by a 3 hr i.v. infusion (2 x 10(5) iu/kg). One hr after intrathecal administration, interferon titers in the CSF were 1000-2000 iu/ml, and declined to less than 10 iu/ml during the first 24 hr. At the same time only minute amounts of HuIFN-beta were found in the sera of these animals. After i.m. administration, 5 iu/ml were found on average in the serum during the first 8 hr, with no interferon titer ever exceeding 10 iu/ml. No antiviral activity was found 24 hr after HuIFN-beta injection. HuIFN-beta injected i.v. resulted in serum interferon titers of approx. 300 iu/ml 30 min after administration, which then decreased to about 5 iu/ml during the first 8 hr 24 hr after injection no antiviral activity was detectable. Daily HuIFN-beta i.v. administrations over a period of four days did not affect the interferon clearance from the circulation. During HuIFN-beta infusion, constant HuIFN-beta titers of about 80 iu/ml were observed which then decreased during the following 5 hr to 2 iu/ml. The choice between a single i.v. injection, resulting in high serum titers for a few minutes, and a 3 hr infusion, resulting in an about fourfold lower but constant interferon level for over two hr, will depend on the mechanism of interferon action in vivo and on the question as to whether side effects can be diminished by infusion.

Animals↗
PubMed1979

Isolation of replication forks from growing Ehrlich ascites cells.

A procedure is described which permits the large-scale isolation of essentially complete replications forks from the DNA of Ehrlich ascites cells. The whole nuclear DNA is first isolated by a method which involves minimal hydrodynamic shear. The DNA is then degraded by cryolysis, a freeze-thawing procedure, to a size providing the otherwise very labile forked structures with a sufficient resistance against shear forces. Finally, the Y-shaped structures of replicating DNA are separated by nitrocellulose column chromatography. When the newly formed strands of replicating DNA were density-labeled with 5-bromodeoxyuridine the DNA fraction isolated by this procedure banded in isopycnic CsCl gradients at a density expected for Y-shaped molecules with two light-heavy branches and one light-light branch and sedimented significantly faster than the corresponding bulk DNA fraction through neutral sucrose gradients. The forked molecules could be visualized by electron microscopy. The essential step of the procedure is the cryolysis which produces fragments from larger DNA structure essentially at random. When the cryolysis is omitted the forked structures are disrupted within the highly susceptible regions around the branching point.

Animals↗
PubMed1979

Gel chromatography applied to quantitation of components of IgG preparations.

Investigations were undertaken to standardize gel chromatography for quality control of IgG preparations. Great importance was attached to convenience of procedure. With application of the optimal type ofgel, Ultrogel AcA 34, the influence on quality of separation of various experimental parameters such as pH and ionic strength of the eluant, volume and concentration of the sample, flow rate and direction, and temperature were tested. Various methods of evaluation were applied. According to the demands, quantitation of components may be performed by collection of fractions and determination of peak volumes and protein concentrations, by determination of peak areas on absorbance recordings, or automatically by use of an integrator or a computer. The results obtained by gel chromatography are discussed and compared with the results of analytical ultracentrifugation.

Chromatography, Gel↗