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C E Hack

Publications and source records attributed to C E Hack.

341 records · Page 19Linked to original sources
PubMed1987

Inactivation of C-1 inhibitor by proteases: demonstration by a monoclonal antibody of a neodeterminant on inactivated, non-complexed C-1 inhibitor.

Monoclonal antibodies were raised against kallikrein-C-1 inhibitor and factor XIIa-C-1 inhibitor complexes. One of the monoclonal antibodies (KII) appeared to react predominantly with C-1 inhibitor complexes in an ELISA. However, the apparent binding of KII to C-1 inhibitor complexes was probably due to the presence of proteolytically inactivated C-1 inhibitor in the complex mixture used for the coating:KII did not bind either kallikrein-C-1 inhibitor or factor XIIa-C-1 inhibitor complexes generated in plasma by dextran sulphate. SDS-PAGE analysis of C-1 inhibitor incubated with proteases revealed that KII-reactive C-1 inhibitor has a lower molecular weight than native C-1 inhibitor. We propose that the determinant that reacts with KII is exposed after cleavage of C-1 inhibitor in its reactive site. The monoclonal antibody KII will enable us to study the inactivation of C-1 inhibitor in human inflammatory disease.

Antibodies, Monoclonal↗
PubMed1987

Formation of C3-IgG complexes in serum by aggregated IgG and by non-immunoglobulin activators of complement.

We studied the generation of C3-IgG complexes during the activation of C3 in serum by aggregated human IgG (AHG), zymosan or cobra venom factor (CVF). C3-IgG complexes were detected by specific radioimmunoassays: samples to be tested were incubated with anti-IgG Sepharose, and complexes that had bound to the Sepharose were detected by incubation with either 125I-anti-C3c or 125I-anti-C3d, g. Incubation of serum with as little as 6 micrograms AHG per ml, for 30 min at 37 degrees, resulted in the generation of C3-IgG complexes. When serum was incubated with zymosan or CVF, C3-IgG complexes were also generated. AHG appeared to be more effective in the generation of C3-IgG complexes than CVF. We calculated that AHG (2 mg/ml) caused about 36% of the C3 to be fixed to IgG, CVF (400 micrograms/ml) about 14%. Finally, the presence of C3 fixed to IgG in serum incubated with CVF was demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting. This study indicates that the formation of C3-IgG complexes in serum is not only induced by immune complexes but also by non-immunoglobulin activators. Therefore, C3-IgG complexes might be considered as complement activation products, and their detection in patients' samples should not be considered as conclusive evidence for the presence of immune complexes.

Antigen-Antibody Complex↗
PubMed1986

Influence of C3 level on the determination of C3d in plasma and synovial fluid by radial immunodiffusion.

The influence of C3 levels on the determination of C3d in plasma and synovial fluid by radial immunodiffusion was investigated. In the method used, C3 is precipitated by 11% polyethylene glycol (PEG), and C3d is measured in the supernatant. In 51 healthy donors, a weak though significant correlation between C3 and C3d levels was found. The mean concentration of C3d was 1.6% of that in aged serum from healthy donors. So, small amounts of C3 (i.e., 1-2% of the normal plasma level) in the 11% PEG supernatants may contribute significantly to the C3d levels measured. A radioimmunoassay that detects C3, C3b, iC3b and C3c was used to measure C3 levels in the PEG supernatants. In PEG supernatants of 4 plasma samples, 0.3-0.6% of the C3 level in normal plasma was found, whereas in those of 2 synovial fluids much higher levels were found (4-10% of the normal plasma level). When purified 125I-labeled antibodies against C3c were added to the gel of the radial immunodiffusion, C3c antigen was detected in the precipitation rings obtained with all PEG supernatants of plasma samples from patients. Therefore, the quantitative contribution of C3 to the precipitation rings in the C3d radial immunodiffusion was analyzed after the addition of an excess of anti-C3c antibodies to the gel. No effect on the size of the C3d-precipitation rings obtained with plasma samples from patients was observed. However, the C3d precipitation rings obtained with synovial fluids were significantly smaller when the gel used in the radial immunodiffusion contained an excess of anti-C3c antibodies together with the anti-C3d serum. We conclude that it is necessary to add an excess of anti-C3c antibodies to the gel used for the radial immunodiffusion, for the determination of C3d levels in synovial fluid. An antiserum against human C3b, which contains both anti-C3c and anti-C3d antibodies, can be used for this purpose.

Blood Donors↗
PubMed1986

The IgG detected in the C1q solid-phase immune-complex assay is not always of immune-complex nature.

The properties of the solid-phase C1q immune-complex assay as well as the nature of the IgG detected by this assay in patients' sera were investigated. Aggregated IgG was used as a model for immune complexes. Aggregated IgG bound to solid-phase C1q was detected by 125I-anti-IgG. Fluid-phase C1q (either in normal human serum or purified) neither inhibited the binding of aggregated IgG to solid-phase C1q nor dissociated bound aggregated IgG from the solid-phase C1q. Therefore, we concluded that the solid-phase C1q has a higher affinity for aggregated IgG than the fluid-phase C1q, probably because of the polymerization of the solid-phase C1q. To get more insight into the nature of the IgG detected by the C1q solid-phase assay in patients' sera, we investigated whether C4 and/or C3 were present on it. With the use of 125I-anti-C4 and 125I-anti-C3 instead of 125I-anti-IgG, C4 and C3, respectively, were easily detected on the aggregated IgG that had bound to the solid-phase C1q. The lower limit of detection of these assays was 30 micrograms aggregated IgG/ml of normal human serum. Sera of patients suffering from rheumatoid arthritis and systemic lupus erythematosus were tested with these assays and, despite positive results with 125I-anti-IgG, no positive results were obtained with either 125I-anti-C4 or 125I-anti-C3. So, on the IgG detected by the C1q solid-phase assay in patients' sera, neither C4 nor C3 are present. Furthermore, in five of the six sera tested, this IgG sedimented as monomeric IgG. Therefore, it seems unjustified to refer to this IgG as circulating immune complexes.

Antigen-Antibody Complex↗
PubMed1986

Chronic idiopathic and secondary neutropenia: clinical and serological investigations.

Clinical data on 49 patients with chronic idiopathic neutropenia (CIN) and 42 patients with neutropenia secondary to a well-defined immunological disorder (SN) were collected and related to serological parameters. In 47% of the patients with CIN and 53% of those with SN, a positive direct immunofluorescence test was obtained with granulocytes from the patients. In the sera from the patients in the two groups, antibodies against donor granulocytes were detected by the indirect immunofluorescence test, the leucoagglutination test and/or the granulocytotoxicity test in 15%, 19% and 15%, respectively. The results of the above tests could not be correlated with any clinical or haematological parameter. Immune complexes in the serum were detected by the 125I-Clq-binding test in 29% of patients with CIN and in 58% of those with SN. The presence of serum immune complexes correlated well with the existence of a low neutrophil count, but not with the presence of recurrent infections, with bone-marrow abnormalities, or with positive reactions in other serological tests. The sera of eight out of 14 patients with CIN and seven out of 12 patients with SN had inhibitory activity for myeloid colony formation in vitro (CFU-GM). This CFU-GM inhibitory activity was correlated with the presence of recurrent infections and with hypoplasia of the myeloid compartment of the bone marrow, but not with positive reactions in other tests. We conclude that the 125I-Clq-binding test probably detects circulating immune complexes that induce a shift neutropenia, whereas serum activity inhibitory for CFU-GM possibly relates to clinically more serious forms of neutropenia. The significance of neutrophil-bound Ig and granulocyte-reactive antibodies in the serum is not clear.

Adolescent↗
PubMed1985

Correlation of disease activity with circulating immune complexes (C1qbA) and complement breakdown products (C3D) in patients with systemic lupus erythematosus. A prospective study.

Most biologic effects of immune complexes are mediated through the activation of the complement system. The relationship between lupus disease activity and the presence of C3 breakdown products (C3d) and circulating immune complexes (CIC) as demonstrated with the C1q binding assay (C1qbA), was evaluated. Nearly all 13 systemic lupus erythematosus (SLE) patients had a stable disease course in this prospective study, nevertheless, in each patient the profiles of the serologic parameters were quite different. Despite the small number of investigated patients (13), it is concluded that irrespective of the disease activity, the serologic parameters could be either positive or negative. No relationship could be obtained between disease activity and the presence of C3d and/or CIC. Nor was there any evidence that the presence of CIC would indicate increased levels of C3 breakdown products (C3d). This observation argues against a pathogenetic significance of CIC detected by the C1qbA in SLE. In conclusion, the supposed link between the presence of CIC, consumption and activation of the complement system, and the activity of SLE needs further study.

Adult↗
PubMed1985

Low molecular weight C1q in systemic lupus erythematosus.

In sera of patients suffering from an exacerbation of systemic lupus erythematosus (SLE), increased amounts of abnormal C1q were detected, contrasting with decreased or even undetectable levels of normal C1q in these sera. When analyzed immunochemically by double immunodiffusion, this low m.w. C1q (LMW-C1q) appeared to be identical with the defective C1q in serum of individuals with an inherited, homozygous inability to produce functional plasma C1q. These persons show a tendency to develop SLE-like syndromes. Like the genetically defective C1q, the abnormal C1q molecule in SLE sera was hemolytically inactive, did not incorporate in C1, was found in the supernatant of euglobulin-precipitated serum, and appeared in the break-through fraction of a cation-exchange column. Sucrose gradients and gel filtration analyses supported the putative identity of the molecules. SDS-PAGE and immunoblots revealed the presence of subunits that reacted with antibodies against C1q and confirmed the C1q-like nature of LMW-C1q. Low levels of LMW-C1q were also detected in serum and plasma of normal individuals. A radial immunodiffusion technique was used to measure LMW-C1q in the serum of 54 patients. Although these patients were not selected for parameters of disease activity, their levels of LMW-C1q were significantly higher than those of normal individuals and children with decreased C3 levels due to acute glomerulonephritis.

Centrifugation, Density Gradient↗
PubMed1984

Influence of ionic strength, EDTA concentration, endogenous C1q and polyanions on the 125I-C1q-binding test.

Several parameters of the 125I-C1q-binding test were investigated: ionic strength, pH, concentration of EDTA, influence of serum C1q and the possibility of interference by polyanions. Lowering the ionic strength of the borate buffer resulted in increased precipitation of 125I-C1q in normal human serum. This increase was dependent on the presence of serum proteins, probably immunoglobulins. When the concentration of the EDTA was decreased, increased precipitation of 125I-C1q in normal human serum was also observed. This was prevented by adding NaCl to the EDTA solution. However at very low concentrations of EDTA (too low to chelate all calcium ions in the serum), increased precipitation of 125I-C1q in normal human serum was observed even in the presence of added NaCl. Addition of purified C1q to sera from patients with very low C1q levels had varying effects on the results of the C1q-binding test: (a) it decreased the C1q-binding activity of some sera, probably by competition with 125I-C1q for binding sites on the immune complexes; (b) it increased the C1q-binding activity of other sera, probably by enhancing the precipitation of immune complexes as a consequence of the cross-linking effect of C1q; or (c) it had no influence, possibly due to the opposite effects of (a) and (b). The addition of dextran sulphate resulted in a dose-dependent increase in the 125I-C1q-binding activity of normal human serum. This effect was dependent on the interaction of dextran sulphate with either C1q or low-density lipoproteins and was prevented by addition of polybrene to the assay. However, addition of polybrene to sera with a high C1q-binding activity scarcely influenced binding activity.

Antigen-Antibody Complex↗
PubMed1984

Lack of activation of C1, despite circulating immune complexes detected by two C1q methods, in patients with rheumatoid arthritis.

The activation of C1 by circulating immune complexes in patients with rheumatoid arthritis was investigated. C1rC1s(C1-In)2 complexes in EDTA-plasma, reflecting C1 activation in vivo, were slightly raised in 35 of 57 patients with rheumatoid arthritis, though most patients had elevated levels of circulating immune complexes as measured with either the 125I-C1q binding test or the C1q solid phase assay. The activation of C1 by circulating immune complexes in vitro was investigated by measuring the generation of C1rC1s(C1-In)2 complexes during 60 minutes at 37 degrees C in diluted recalcified EDTA-plasma. In 16 of the 57 patients, a slightly increased C1 activation in vitro was observed. These patients tended to have high levels of circulating immune complexes. However, the majority of the patients with high levels of circulating immune complexes showed a normal C1 activation in vitro. Therefore, it was concluded that measurement of circulating immune complexes by either of the two C1q methods in patients with rheumatoid arthritis does not imply that these circulating immune complexes are able to activate C1.

Aged↗
PubMed1984

Immunisation of guinea-pigs with circulating immune complexes from patients with rheumatoid arthritis.

Sixteen guinea-pigs were immunised with immune complexes isolated from serum of nine patients with rheumatoid arthritis. The resulting antisera were analysed by radioimmunoassays. All guinea-pig sera were extensively absorbed with normal human serum. After this absorption eight guinea-pig sera contained antibodies specific for immune complexes isolated from the sera of three patients. One of these antisera reacted not only with immune complexes (and serum) from the corresponding patient but also with immune complexes (and sera) from other patients with rheumatoid arthritis. The antigen(s) to which the guinea-pig antibodies were directed sedimented as IgM, and they bound to IgG Sepharose. Therefore the guinea-pig sera were absorbed with IgM-rheumatoid factors isolated from the serum of the corresponding patient. After this absorption, the guinea-pig sera had lost their reactivity with immune complexes. We conclude that these antisera did not detect an exogenous antigen in immune complexes from patients with rheumatoid arthritis. The positive reactions found were due to antibodies specific for (idiotypic?) antigenic determinants on IgM-rheumatoid factors.

Animals↗
PubMed1984

SLE like syndrome and functional deficiency of C1q in members of a large family.

Two sisters and a brother from one family are described whose sera were deficient in haemolytic complement function. This defect was restored by addition of purified C1q. In their sera, C1q like material was found, whereas C1r and C1s were normal or increased in concentration, as were the other complement components tested. All three had suffered from glomerulonephritis during childhood. A renal biopsy in the brother recently disclosed a membranous glomerulopathy stage 1; otherwise, he is apparently healthy. In both sisters, a systemic lupus erythematosus like disease became manifest at the age of 20 and 23, respectively, resulting in the death of one of them. In the serum of these three family members, the C1q like material was antigenically deficient compared with normal C1q and had, on sucrose gradient analysis, a molecular weight of approximately 65,000 daltons. It did not bind to C1r and C1s. Binding of the dysfunctional C1q to aggregated human gammaglobulin could be demonstrated. On double immunodiffusion analysis, the abnormal C1q was identical with reduced and alkylated C1q. The possible structure of the abnormal C1q molecule is discussed.

Adult↗
PubMed1983

Interference of IgG, IgG aggregates and immune complexes in tests for platelet autoantibodies.

Three techniques, based on the antiglobulin principle, used for the detection of autoantibodies against platelets, were compared; the antiglobulin consumption assay (QACA), the platelet radioactive antiglobulin test (PRAT) and the platelet suspension immunofluorescence test (PSIFT). Upon incubation of normal donor platelets with purified IgG, in concentrations higher than that in serum, an increased amount of platelet-associated IgG was demonstrated only in the QACA. Upon incubation with aggregated IgG, all three tests became positive, but the PSIFT only with high concentrations of aggregates. Binding of soluble C1q-binding immune complexes (IC), which consisted of tetanus toxoid and IgG antitetanus antibodies (TaT) to normal donor platelets, was only detectable in the QACA. However, a positive result was obtained in all three tests with platelets incubated with soluble DNA-IgG-antiDNA antibodies (DaD) IC. Fixation of the platelets with paraformaldehyde prevented the binding and the detection of the DaD-IC, but not of IgG, aggregated IgG or TaT-IC. Eluates from platelets incubated with aggregated IgG, TaT- or DaD-IC did not react with normal donor platelets in the three techniques, in contrast to eluates from platelets sensitized with platelet antibodies.

Antibodies, Antinuclear↗
PubMed1981

Polyethylene glycol enhances the binding of C1q to circulating immune complexes.

By radioimmunoassay we measured the amount of endogenous C1q that was precipitated by polyethylene glycol (PEG) under the conditions of the 125I-C1q-binding test (C1q-BT). We found a linear correlation between the percentage endogenous C1q that was precipitated and the 125I-C1q-binding activity (C1q-BA). We concluded that the 125I-C1q behaves like the endogenous C1q. To detect circulating immune complexes (CIC) which had already bound C1q, human sera were added to tubes coated with anti-C1q. Under the conditions used, no C1q-bearing CIC were detected. In addition, 7 sera from patients with high C1q-BA were analyzed by sucrose-gradient ultracentrifugation. No C1q was found in the fast sedimenting fractions, although C1q-BA was detected in these fractions. With IgG-coated tubes we observed that PEG enhanced the binding of 125I-C1q as well as endogenous C1q to aggregated and monomeric IgG. PEG also enhanced the binding of CIC to C1q-coated tubes. The results suggest that CIC detected by the C1q-BT do not bear C1q in significant amounts in the circulation and that these CIC become detectable only in the presence of PEG.

Animals↗
PubMed1981

A C1-inhibitor-complex assay (INCA): a method to detect C1 activation in vitro and in vivo.

A radioimmunoassay (the C1-inhibitor-complex assay, INCA) is described for the detection of complexes that are composed of at least C1s and C1-inhibitor. This INCA is based on demonstrating that C1s and C1-inhibitor (C1-In) are linked: after an incubation with anti-C1s-Sepharose, bound C1sC1-In complexes are detected by 125I-anti-C1-In. C1sC1-In complexes were prepared by the addition of a slight excess of C1s to normal human serum (NHS). As little as 2 ng C1-In bound to C1s was detected. Additional free C1s in serum hardly influenced the detection of C1sC1-In complexes. Complexes presumably composed of C1rC1s(C1-In)2 were generated by the addition of aggregated IgG to NHS. This generation was inhibited by lowering the temperature to 0 degrees C, and by EDTA, and depended on the concentration of aggregated IgG. These complexes had a sedimentation value of approximately 9S. Complexes of C1s and C1-In were also generated in NHS by the addition of DNP-albumin and protein A, but not by zymosan. The INCA was applied to blood samples from normal donors and patients. Sixteen out of 19 samples from patients with acute glomerulonephritis contained increased amounts of C1rC1s(C1-In)2 complexes as compared with the amounts in blood samples from normal donors. The INCA provides a useful tool to assess the activation of C1 in the presence of C1-In, both in vitro and in vivo.

Complement Activation↗
PubMedDate not supplied

Interplay of complement and cytokines in the pathogenesis of septic shock.

Sepsis is a clinical syndrome that is usually induced by bacterial infections. It is generally assumed that the syndrome results from an excessive triggering of endogenous inflammatory mediators by the invading microorganisms. These mediators include substances released by activated monocytes, macrophages, endothelial cells and neutrophils such as cytokines, reactive oxygen species and proteases, as well as activation products of coagulation, fibrinolysis, contact and complement systems. Recent studies have suggested that cytokines and complement activation products may have overlapping biological activities. In addition, multiple interactions in vitro as well as in vivo between cytokines and complement have been described. Here we will review some of these recent studies and will discuss their relevance for the pathogenesis of sepsis and septic shock.

Animals↗