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Biomedical subjects

C E Hack

Publications and source records attributed to C E Hack.

At least 325 records · Page 18Linked to original sources
PubMed1989

Elevated plasma levels of the anaphylatoxins C3a and C4a are associated with a fatal outcome in sepsis.

PURPOSE AND PATIENTS AND METHODS: Both complement and contact system of coagulation have been implicated in the pathophysiology of sepsis. We therefore measured levels of the complement activation products C1-C1-inhibitor complexes and C3a in serial plasma samples (obtained every six hours) from 48 patients with clinically suspected sepsis, and related these levels to the clinical outcome. C4a was also measured in samples obtained on admission. RESULTS: C3a levels were elevated in 47 patients at least once during the observation period. These levels appeared to be considerably higher in patients who died than in patients who survived. This difference was found for the levels on admission (p = 0.0003), as well as for the highest (p = 0.0010) and the lowest (p less than 0.0001) levels encountered in each patient. The mortality in patients with plasma C3a levels of 13 nmol/liter or less on admission (27 patients) was 33 percent, compared with 86 percent in patients with levels of 14 nmol/liter or more. Patients with septic shock had significantly higher C3a levels than normotensive patients (p values between 0.046 and 0.004). No significant differences in C3a were found between patients who had respiratory distress syndrome and those who did not. C4a levels in plasma samples obtained on admission were elevated in 43 patients. These levels correlated very significantly with C3a levels (p less than 0.0001), and showed similar associations with a fatal outcome. C1-C1-inhibitor complexes were elevated in 23 patients at least once during the observation period. These patients had significantly higher levels of C4a and C3a than patients with normal amounts of C1-C1-inhibitor complexes. Patients who died had higher levels of C1-C1-inhibitor complexes than patients who survived. However, this difference was not significant. CONCLUSION: On the basis of our results, we propose that activation of the complement system via the classical pathway is involved in the development of fatal complications in sepsis.

Adolescent↗
PubMed1989

Proteolytic inactivation of plasma C1- inhibitor in sepsis.

Activation of both the complement system and the contact system of intrinsic coagulation is implicated in the pathophysiology of sepsis. Because C1 inhibitor (C1-Inh) regulates the activation of both cascade systems, we studied the characteristics of plasma C1-Inh in 48 patients with severe sepsis on admission to the Intensive Care Unit at the Free University of Amsterdam. The ratio between the level of functional and antigenic C1-Inh (functional index) was significantly reduced in the patients with sepsis compared with healthy volunteers (P = 0.004). The assessment of modified (cleaved), inactive C1-Inh (iC1-Inh), and complexed forms of C1-Inh (nonfunctional C1-Inh species) revealed that the reduced functional index was mainly due to the presence of iC1-Inh. On SDS-PAGE, iC1-Inh species migrated with a lower apparent molecular weight (Mr 98,000, 91,000, and 86,000) than native C1-Inh (Mr 110,000). Elevated iC1-Inh levels (greater than or equal to 0.13 microM) were found in 81% of all patients, sometimes up to 1.6 microM. Levels of iC1-Inh on admission appeared to be of prognostic value: iC1-Inh was higher in 27 patients who died than in 21 patients who survived (P = 0.003). The mortality in 15 patients with iC1-Inh levels up to 0.2 microM was 27%, but in 12 patients with plasma iC1-Inh exceeding 0.44 microM, the mortality was 83%. The overall mortality in the patients with sepsis was 56%. We propose that the cleavage of C1-Inh in patients with sepsis reflects processes that play a major role in the development of fatal complications during sepsis.

Complement C1 Inactivator Proteins↗
PubMed1989

Wegener's granulomatosis autoantibodies identify a novel diisopropylfluorophosphate-binding protein in the lysosomes of normal human neutrophils.

Anti-neutrophil cytoplasmic autoantibodies (ANCA) specifically associated with Wegener's granulomatosis were found to be directed against a saline-soluble glycoprotein triplet that migrates on SDS gels as distinct bands of Mr 29,000, 30,500, and 32,000 and is present in the azurophilic granules. This antigen was specifically recognized by all cytoplasmic-staining (C)-ANCA-positive sera from patients with Wegener's disease. C-ANCA antigen bound [3H]diisopropylfluorophosphate, which indicates that it is a serine protease, but it could clearly be distinguished from the serine proteases elastase and cathepsin G. Stimulation of cytochalasin B-treated neutrophils with FMLP induced release of C-ANCA antigen. This indicates that in vivo C-ANCA might interact with the C-ANCA antigen after its release upon inflammatory stimulation. We further demonstrate that in some perinuclear staining (P-ANCA) patients' sera autoantibodies against other myeloid lysosomal enzymes can be detected, such as antimyeloperoxidase and antielastase. C-ANCA and P-ANCA thus represent a novel class of autoantibodies directed against myeloid lysosomal enzymes. The originally described Wegener-specific C-ANCA show an apparently uniform specificity for the 29,000 serine protease. In contrast, P-ANCA may recognize myeloperoxidase as well as elastase and/or other antigens.

Antibodies, Monoclonal↗
PubMed1989

Increased plasma levels of interleukin-6 in sepsis.

Interleukin-6 (IL-6) is likely to be an important mediator of the inflammatory response. We measured levels of this cytokine in plasma samples from 37 patients with sepsis or septic shock obtained at the time of admission to the intensive care unit and related these levels to hemodynamic and biochemical parameters as well as to clinical outcome. In 32 of the 37 patients, increased levels of IL-6 were found, occasionally up to 7,500 times the normal level. The highest IL-6 levels were encountered in patients who suffered from septic shock (P value of the difference between patients with and without shock less than .0001). In addition, IL-6 significantly correlated with plasma lactate (P less than .0001), heart rate (P = .05) and, inversely, with mean arterial pressure (P = .01) and platelet counts (P = .0002). Significant correlations of IL-6 with the anaphylatoxins C3a (P = .0001) and C4a (P = .0002) and with the main inhibitor of the classical pathway of complement, C1-inhibitor (inverse correlation, P = .05), were also observed. IL-6 on admission appeared to be of prognostic significance: levels were higher in septic patients who subsequently died than in those who survived (P = .0003), in particular when only patients with septic shock were considered (P less than .0001). All nine septic patients with levels of less than 40 U/mL on admission survived, whereas 89% of the nine patients with levels exceeding 7,500 U/mL died. These data provide evidence for a role of IL-6 in the pathophysiology of septic shock. Further studies are needed to reveal whether IL-6 in sepsis is directly involved in mediating lethal complications or whether it is to be considered as an "alarm hormone" that reflects endothelial cell injury probably mediated by the anaphylatoxines.

Bacterial Infections↗
PubMed1988

Disruption of the internal thioester bond in the third component of complement (C3) results in the exposure of neodeterminants also present on activation products of C3. An analysis with monoclonal antibodies.

Hydrolysis of the internal thioester bond in native C3 is thought to be a key event in initiating the alternative pathway of C activation, because the resulting C3(H2O) acquires "C3b-like" properties. Therefore, disruption of the internal thioester bond is probably accompanied by conformational changes in the C3 molecule. In this study, we demonstrate that such conformational changes indeed occur; 7 of the 19 mAb raised against C3 or C3 activation products recognized epitopes exposed on C3(H2O) but not on native C3. One of these epitopes is located on the C3a part, three on the C3c part, and another three on the C3d,g part. Because the 7 mAb bound equally well to C3 incubated either with MgCl2 or with methylamine (which primarily disrupts the thioester), the conformational changes detected by the mAb apparently occur after disruption of the thioester. Furthermore, the epitopes were also present on the corresponding C3 activation products. Immunoblotting experiments revealed that the epitopes for the three anti-C3d,g mAb were located on the C3d part, C-terminal to the thioester. The epitopes for 2 of the 3 anti-C3c mAb were located on the C-terminal alpha-chain fragment of C3c. Thus, this study provides immunochemical evidence for the biologic resemblance between C3(H2O) and C3 activation products. Implications of these findings for the activation process of C3 are discussed.

Animals↗
PubMed1988

Expression of functional human C1 inhibitor in COS cells.

Full length human C1 inhibitor cDNA was cloned into a vector suitable for transient expression in COS-1 cells. Transfected COS cells secreted an immunoreactive protein of Mr approximately 110,000 that appeared to be functionally equivalent to the plasma-derived protein as established by the following criteria: 1) ability to form sodium dodecyl sulfate-stable complexes with C1s, factor XIIa, and kallikrein; 2) inhibition of C1s-mediated C4 consumption; and 3) susceptibility to inactivation by the nontarget proteinase elastase. Quantitation of secreted recombinant C1 inhibitor by radioimmunoassay indicated that 72 h after transfection the level was approximately 2.2 micrograms/ml. Treatment of transfected cells with tunicamycin resulted in secretion of a protein of Mr approximately 90,000 that was also capable of complex formation with C1s.

Animals↗
PubMed1988

A modified competitive inhibition radioimmunoassay for the detection of C3a. Use of 125I-C3 instead of 125I-C3a.

Levels of C3a in plasma are currently measured by a competitive inhibition radioimmunoassay (RIA) in which 125I-C3a is used as a tracer. In this paper, we describe a modification of this RIA: 125I-C3 instead of 125I-C3a is used. The lower limit of detection of this modified RIA is 6 ng of C3a per ml of plasma (i.e. 0.66 nmol/l). This RIA, performed with polyclonal anti-C3a antibodies coupled to a solid phase, appeared to be 30 times more sensitive compared with an RIA in which a monoclonal antibody against C3a is used. In vitro activation of the complement system in serum by aggregated IgG, zymosan, and cobra venom factor resulted in the generation of significant amounts of C3a. Assessment of the C3a levels by the modified RIA in serial plasma samples from patients who underwent cardiopulmonary bypass, yielded results very similar to those described in the literature for the established C3a-RIA. Thus, the modified C3a-RIA offers a convenient alternative for the detection of C3a in plasma samples.

Antibodies, Monoclonal↗
PubMed1988

Complement (C3) metabolism in rheumatoid arthritis in relation to the disease course.

Metabolic turnover studies of the third component of complement, C3, were performed in 23 patients with rheumatoid arthritis (RA) to get a direct insight in the dynamics of complement synthesis and catabolism. Results of these turnover studies were related to the serum level of the total amount of C3 as well as to that of the activation product C3d. A hypercatabolism of C3 was observed in 12 of the 23 patients studied. Six of these 12 patients showed signs of extra-articular RA; only one patient with extra-articular manifestations had a normal catabolism of C3. Decreased serum levels of C3 were not found in any of the patients with a hypercatabolism of C3, indicating that the accelerated turnover was compensated by an increased synthesis. In RA patients levels of the activation product C3d could not correlate with the turnover of C3. However, in selected RA patients without signs of nodules or extra-articular manifestations, they could. Thus, our results indicate that serum levels of C3 and C3d do not reflect C3 metabolism in RA patients. Furthermore, the existence of extra-articular manifestations is accompanied by a more pronounced activation of the complement system.

Arthritis, Rheumatoid↗
PubMed1988

The distortive mechanism for the activation of complement component C1 supported by studies with a monoclonal antibody against the "arms" of C1q.

A mouse monoclonal antibody (IgG1 isotype) against human C1q (MAb 130) is presented that activates C1 in serum through its antigen-binding sites at an optimal molar ratio of 3 MAbs:1 C1q. The antibody does not inhibit binding of C1q to IgG. Experiments with pepsin- and collagenase-digested C1q showed that MAb 130 binds to the fibril-like strands (arms) of C1q, close to the globular heads. Bivalency of MAb 130 was a requirement for C1-activation, but not for binding to C1q. Increasing the segmental flexibility of the intact antibody by reduction and alkylation destroyed its capacity to activate C1. A MAb against the globular heads of C1q completely inhibited C1-activation by aggregated IgG (AHG), but did not prevent activation by MAb 130. C1, reconstituted by adding C1q-stalks that lack the globular heads to C1q-depleted serum was not activated by AHG, whereas activation by MAb 130 was not affected. Activation of serum-C1 by AHG and MAb 130 was inhibited by addition of excess purified C1-inhibitor in a comparable and dose-dependent manner. Sucrose-gradient analysis indicated a predominance of stable complexes of a single C1q-molecule with three MAbs at the optimal activating ratio. When isolated and added to C1q-depleted serum, these complexes activated C1 efficiently. A mechanism for activation by MAb 130 is proposed that supports the "distortive" model of C1-activation.

Antibodies, Monoclonal↗
PubMed1988

Covalent cross-links in oxygen free radical altered human immunoglobulin G.

The damaging effect of an oxygen free radical generating system, i.e. ultraviolet irradiation, on human immunoglobulin G (IgG) was studied. The free radical altered IgG was analysed by a high performance liquid chromatograph equipped with a TSK G 3000 SW-column. Gel filtration of 120 min UV-irradiated IgG resulted in three clearly distinguished peaks corresponding to polymer IgG (MW greater than 500 kD), dimer IgG (MW 300 kD) and monomer IgG (MW 150 kD). Analysis of oxygen free radical altered and aggregated IgG by SDS-PAGE and subsequent silver-staining revealed inter- and intra-molecular reduction (by beta-mercaptoethanol)-resistant cross-links between IgG-molecules were formed. Comparison of amino acid analyses of native IgG with oxygen free radical aggregated polymer IgG showed significant reductions in tyrosine- (7.0%) and histidine- (6.5%) content. These findings suggest that tyrosine and histidine are involved in covalent cross-linking between IgG-molecules caused by oxygen free radicals. These alterations on IgG induced by free radical-activity might render it antigenic, and could initiate the production of rheumatoid factors (RF).

Amino Acids↗
PubMed1988

Quantification of plasma factor XIIa-Cl(-)-inhibitor and kallikrein-Cl(-)-inhibitor complexes in sepsis.

Considerable evidence indicates that activation of the contact system of intrinsic coagulation plays a role in the pathogenesis of septic shock. To monitor contact activation in patients with sepsis, we developed highly sensitive radioimmunoassays (RIAs) for factor XIIa-Cl(-)-inhibitor (Cl(-)-Inh) and kallikrein-Cl(-)-Inh complexes using a monoclonal antibody (MoAb Kok 12) that binds to a neodeterminant exposed on both complexed and cleaved Cl(-)-Inh. Plasma samples were serially collected from 48 patients admitted to the intensive care unit because of severe sepsis. Forty percent of patients on at least one occasion had increased levels of plasma factor XIIa-Cl(-)-Inh (greater than 5 x 10(-4) U/mL) and kallikrein-Cl(-)-Inh (greater than 25 x 10(-4) U/mL), that correlated at a molar ratio of approximately 1:3. Levels of factor XII antigen in plasma and both the highest as well as the levels on admission of plasma factor XIIa-Cl(-)-Inh in 23 patients with septic shock were lower than in 25 normotensive patients (P = .015: factor XII on admission; P = .04: highest factor XIIa-Cl(-)-Inh; P = .01: factor XIIa-Cl(-)-Inh on admission). No significant differences in plasma kallikrein-Cl(-)-Inh or prekallikrein antigen were found between these patients' groups. Elevated Cl(-)-Inh complex levels were measured less frequently in serial samples from patients with septic shock than in those from patients without shock (P less than .0001). Based on these results, we conclude that plasma Cl(-)-Inh complex levels during sepsis may not properly reflect the extent of contact activation.

Adolescent↗
PubMed1987

Detection of activation of the contact system of coagulation in vitro and in vivo: quantitation of activated Hageman factor-C-1-inhibitor and kallikrein-C-1-inhibitor complexes by specific radioimmunoassays.

Radioimmunoassays (RIAs) for the detection of C-1-inhibitor (C-1-Inh) complexed to either kallikrein or activated Hageman factor (factor XIIa) are described. Kallikrein-C-1-Inh or factor XIIa-C-1-Inh complexes were bound to Sepharose to which monospecific antibodies against (pre)kallikrein or factor XII, respectively, were coupled. Bound complexes were subsequently detected by an incubation with affinity purified 125I-labeled antibodies against C-1-Inh. These RIAs were used to detect activation of the contact system of coagulation in vitro and in vivo. Addition of dextran sulfate (DXS) (20 micrograms/ml) to fresh plasma resulted at 37 degrees C in the rapid generation of amidolytic kallikrein activity, which was maximal after 1 to 2 min of incubation and subsequently decreased within a few minutes. The generation of kallikrein activity coincided with the appearance of both kallikrein-C-1-Inh and factor XIIa-C-1-Inh complexes. However, in contrast to kallikrein activity, both types of complexes remained detectable in the incubation mixtures during the incubation period. Experiments with purified kallikrein. C-1-Inh and partly purified beta-factor XIIa, and activation experiments in plasmas deficient in either factor XII or prekallikrein, demonstrated the specificity of both RIAs. The minimal amount of DXS that resulted in the generation of measurable amounts of both types of complexes in plasma was 2-3 micrograms per ml. Similar experiments with kaolin showed that with limiting amounts of activator (1-2 mg/ml), only kallikrein-C-1-Inh complexes were detected in plasma. When larger amounts of kaolin were added to plasma, factor XIIa-C-1-Inh complexes were additionally detected in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Coagulation↗