PubMed Health⌕ Search

Biomedical subjects

C E Hack

Publications and source records attributed to C E Hack.

At least 163 records · Page 9Linked to original sources
PubMed1997

Pretreatment with a 55-kDa tumor necrosis factor receptor-immunoglobulin fusion protein attenuates activation of coagulation, but not of fibrinolysis, during lethal bacteremia in baboons.

Baboons (Papio anubis) receiving a lethal intravenous infusion with live Escherichia coli were pretreated with either a 55-kDa tumor necrosis factor (TNF) receptor-IgG fusion protein (TNFR55:IgG) (n = 4, 4.6 mg/kg) or placebo (n = 4). Neutralization of TNF activity in TNFR55:IgG-treated animals was associated with a complete prevention of mortality and a strong attenuation of coagulation activation as reflected by the plasma concentrations of thrombin-antithrombin III complexes (P < .05). Activation of fibrinolysis was not influenced by TNFR55:IgG (plasma tissue-type plasminogen activator and plasmin-alpha2-antiplasmin complexes), whereas TNFR55:IgG did inhibit the release of plasminogen activator inhibitor type I (P < .05). Furthermore, TNFR55:IgG inhibited neutrophil degranulation (plasma levels of elastase-alpha1-antitrypsin complexes, P < .05) and modestly reduced release of secretory phospholipase A2. These data suggest that endogenous TNF contributes to activation of coagulation, but not to stimulation of fibrinolysis, during severe bacteremia.

Animals↗
PubMed1997

Effects of interferon-alpha (IFN-alpha) administration on leucocytes in healthy humans.

Plasma concentrations of IFN-alpha are increased in several inflammatory conditions. Several lines of evidence indicate that IFN-alpha has anti-inflammatory properties. To study the effects of IFN-alpha on leucocyte subsets and activation and on cytokines, we administered IFN-alpha (rhIFN-alpha2b; 5 x 10(6) U/m2) to eight healthy human subjects in a randomized controlled cross-over study and analysed changes in circulating leucocytes and parameters for neutrophil and monocyte activation. After administration of IFN-alpha, neutrophil counts increased, monocyte counts decreased transiently, whereas the number of lymphocytes, basophils and eosinophils showed a sustained decrease. IFN-alpha administration was also associated with neutrophil activation, reflected in an increase in the plasma concentrations of elastase-alpha1-antitrypsin complexes and lactoferrin. Serum neopterin, a marker for monocyte activation, was significantly increased 10 h after administration of IFN-alpha. IFN-alpha significantly increased plasma concentrations of IL-6, IL-8 and IL-10. Although IL-1 and tumour necrosis factor (TNF) remained undetectable, plasma concentrations of soluble TNF receptors p55 and p75 increased after IFN-alpha administration. We conclude that IFN-alpha induces multiple alterations in the distribution and functional properties of leucocytes. IFN-alpha exerts pro- as well as anti-inflammatory effects within the cytokine network.

Adult↗
PubMed1997

Intrathecal production of interleukin-12 and gamma interferon in patients with bacterial meningitis.

To assess the role of interleukin-12 (IL-12) and gamma interferon (IFN-gamma) in children with bacterial meningitis, bioactive IL-12 (p70) and the inactive subunit p40 and IFN-gamma were measured in serum and cerebrospinal fluid (CSF) from 35 children with bacterial meningitis and 10 control subjects. The production of IFN-gamma is induced by IL-12 with tumor necrosis factor alpha (TNF-alpha) as a costimulator and inhibited by IL-10. CSF concentrations of IL-12 p40 as well as those of IFN-gamma were markedly elevated, whereas IL-12 p70 was hardly detectable. Detectable CSF levels of IFN-gamma correlated positively with IL-12 p40 (r = 0.40, P = 0.02) and TNF-alpha (r = 0.46, P = 0.04) but not with IL-6, IL-8, or IL-10. In contrast to CSF levels of TNF-alpha, IL-12, and IL-10, those of IFN-gamma were significantly higher in patients with pneumococcal meningitis than in children with meningitis caused by Haemophilus influenzae and Neisseria meningitidis, presumably because of a high CSF TNF-alpha/IL-10 ratio in the former. We suggest that IL-12- and TNF-alpha-induced IFN-gamma production may contribute to the natural immunity against microorganisms in the CSF compartment during the acute phase of bacterial meningitis.

Adolescent↗
PubMed1997

Acute lung injury after aortic surgery: the relation between lung and leg microvascular permeability to 111indium-labelled transferrin and circulating mediators.

BACKGROUND: Aortic surgery is a risk factor for acute lung injury and this may relate to ischaemia/reperfusion (I/R) of the lower body and release of inflammatory mediators. The aim of this study was to define the changes in microvascular protein permeability and circulating inflammatory mediators after aortic surgery. METHODS: In 11 consecutive patients who underwent elective aortic surgery microvascular permeability in lung and leg was measured before and a median of 2.8 hours after completion of surgery using 111indium (In)-labelled transferrin and 99mtechnetium (Tc)-labelled red blood cells, yielding a protein leak index (PLI) that is specific for protein permeability. Circulating leucocyte counts and levels of inflammatory mediators were determined. RESULTS: In the lung the PLI rose from a median of 0.6 (range -0.5 to 2.2) x 10(-3)/min before surgery to 5.4 (-2.3 to 33.5) x 10(-3)/min after surgery, and in the leg from 0.3 (-1.6 to 1.7) x 10(-3)/min to 5.0 (1.0 to 27.8) x 10(-3)/min. The increase in PLI in the lung was related to that in the leg. Levels of activated complement C3a and tumour necrosis factor-alpha did not change, but levels of interleukin (IL)-6, IL-8 and elastase-alpha 1-antitrypsin increased. After surgery there was slight neutrophilia and the leucocyte counts were inversely related to the IL-8 level. The rise in lung but not in leg PLI was greatest in patients with the highest IL-8 levels and the lowest leucocyte counts. CONCLUSIONS: Early after aortic surgery microvascular protein permeability increases in the leg and lung. Leg I/R injury may result in neutrophil activation and release of IL-8, which may induce neutrophil sequestration and subsequently increased pulmonary microvascular permeability. These findings may help to explain the occurrence of acute lung injury after I/R in man.

Adult↗
PubMed1997

Effects of heparin therapy on fibrinolysis in patients with pulmonary embolism.

Previous investigations suggested that heparin administration to humans enhances the tissue type plasminogen activator (tPA) levels in blood, but it remains uncertain whether this effect induces fibrinolysis. We studied the effect of therapeutic levels of heparinization on plasma markers for fibrinolysis in patients suspected of pulmonary embolism (PE). Blood samples were taken from 49 consecutive patients; 28 had confirmed PE, 21 had PE excluded. On admission, the plasma levels of plasmin-alpha 2antiplasmin complexes and D-dimer were significantly higher in the patient group with PE compared to those in whom PE was excluded. After heparinization the tPA levels increased in both groups, showing that this effect was not dependent on the initial level of activity of fibrinolysis. In spite of this increment in tPA levels, the concentrations of plasmin-alpha 2antiplasmin complexes and D-dimer decreased. In conclusion, although heparinization in patients with or without pulmonary embolism does lead to elevated tPA:Ag levels, this is not accompanied by enhanced fibrinolysis.

Adult↗
PubMed1997

Antithrombin acts as a negative acute phase protein as established with studies on HepG2 cells and in baboons.

Patients with sepsis or after major surgery have decreased plasma levels of the anticoagulant protein antithrombin. In such patients elevated levels of interleukin-6 (IL-6) are present and this interleukin is known to induce positive and negative acute phase responses. To investigate the possibility that antithrombin acts as a negative acute phase response-protein we performed studies on the human hepatoma cell line HepG2 in vitro and baboons in vivo. HepG2 cells were treated with recombinant human IL-6, IL-1beta, or combinations of the latter two, and tested for production of antithrombin, fibrinogen and prealbumin (transthyretin). This treatment resulted in a dose dependent increase in fibrinogen concentration (with a maximum effect of 2.8-2.9-fold) and a dose dependent decrease in prealbumin (with a maximum effect of 0.6-0.7-fold) and antithrombin concentrations (with a maximum effect of 0.6-0.8-fold). Simultaneous treatment of the HepG2 cells with IL-6 (1,000 pg/ml or 2,500 pg/ml) and IL-1beta (25 pg/ml), provided more extensively decreased prealbumin (0.8 and 0.6-fold, respectively) and antithrombin concentration (0.7 and 0.6-fold, respectively) compared to the single interleukin treatment at these concentrations. Baboons treated with 2 microg IL-6 x kg body-weight(-1) x day(-1) showed increased plasma CRP levels (59-fold, p <0.05) and decreased prealbumin (0.9-fold, p <0.05) and antithrombin (0.8-fold, p <0.05) plasma levels, without evidence for coagulation activation. Our results indicate that antithrombin acts as a negative acute phase protein, which may contribute to the decreased antithrombin plasma levels observed after major surgery or in sepsis.

Acute-Phase Reaction↗
PubMed1997

Systemic coagulation and fibrinolysis in patients with or at risk for the adult respiratory distress syndrome.

The authors sought to evaluate the pathogenetic and prognostic role of a procoagulant and hypofibrinolytic state in the adult respiratory distress syndrome (ARDS). Twenty-two consecutive patients admitted to the intensive care unit (ICU) for respiratory monitoring (n = 2) or mechanical ventilation (n = 20) were studied, of whom 13 had ARDS and 9 were at risk for the syndrome. Plasma levels of thrombin-antithrombin III complexes (TAT), the plasmin-alpha2-antiplasmin complexes (PAP), tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1) were measured within 48 h after admission, together with respiratory variables allowing computation of the lung injury score (LIS), and pulmonary microvascular permeability [67Gallium-transferrin pulmonary leak index (PLI)], as measures of pulmonary dysfunction. Blood was also sampled 6-hourly until 2 days after admission. The LIS and PLI were higher in ARDS than at risk patients, in the presence of similar systemic morbidity and mortality. TAT complexes were elevated in a minority of patients of both groups, whereas the PAP, tPA and PAI levels were elevated above normal in the majority of ARDS and at risk patients, but groups did not differ. Neither circulating coagulation nor fibrinolysis variables correlated to either LIS or PLI. Furthermore, the course of haemostatic variables did not relate to outcome. These data indicate that systemic activation of coagulation and impaired fibrinolysis do not play a major role in ARDS development and outcome in patients with acute lung injury.

Adult↗
PubMed1996

Effect of factor Xa inhibitors on thrombin formation and complement and neutrophil activation during in vitro extracorporeal circulation.

BACKGROUND: Even when large doses of heparin are administered during cardiopulmonary bypass, thrombin is produced. Thrombin is a powerful protease that is associated with the thrombotic and bleeding complications of open heart surgery and is produced by cleavage of prothrombin by factor Xa. This study assessed the ability of a specific inhibitor of factor Xa, recombinant tick anticoagulant peptide (rTAP), alone or in combination with standard heparin and a low-molecular-weight heparin, enoxaparin, to suppress thrombin formation and activity during in vitro extracorporeal circulation. METHODS AND RESULTS: Fresh, anticoagulated human blood was recirculated for 2 hours in an extracorporeal membrane oxygenator perfusion circuit at 37 degrees C. Four anticoagulant protocols were evaluated; porcine heparin (3.75 U/mL); enoxaparin (17.5 U/mL); rTAP (4 mumol/L); and porcine heparin plus rTAP (2 mumol/L). Blood samples were obtained for analysis from the donor, after anticoagulation, and after 5, 30, 60, and 120 minutes of recirculation. There were no significant differences between groups in platelet count, response to adenosine diphosphate, or prothrombin fragment (F1.2) production. rTAP plus heparin reduced beta-thromboglobulin release; fibrinopeptide A concentrations were significantly higher with rTAP alone. Enoxaparin strongly and significantly inhibited complement C5b9 production and neutrophil elastase release and was associated with significantly increased concentrations of C1-C1 inhibitor and kallikrein-C1 inhibitor complexes. CONCLUSIONS: rTAP does not reduce thrombin formation or activity during in vitro extracorporeal circulation. Enoxaparin markedly inhibits formation of the complement membrane attack complex and neutrophil elastase release, possibly by accelerating C1 inhibitor activity.

Adenosine Diphosphate↗
PubMed1996

Tumor necrosis factor-alpha induces activation of coagulation and fibrinolysis in baboons through an exclusive effect on the p55 receptor.

Tumor necrosis factor-alpha (TNF-alpha) can bind to two distinct transmembrane receptors, the p55 and p75 TNF receptors. We compared the capability of two mutant TNF proteins with exclusive affinity for the p55 or p75 TNF receptor with that of wild type TNF, to activate the hemostatic mechanism in baboons. Both activation of the coagulation system, monitored by the plasma levels of thrombin-antithrombin III complexes, and activation of the fibrinolytic system (plasma levels of tissue-type plasminogen activator, and plasminogen activator inhibitor type I), were of similar magnitude after intravenous injection of wild type TNF or the TNF mutant with affinity only for the p55 receptor. Likewise, wild type TNF and the TNF p55 specific mutant were equally potent in inducing neutrophil degranulation (plasma levels of elastase-alpha 1-antitrypsin complexes). Wild type TNF tended to be a more potent inducer of secretory phospholipase A2 release than the p55 specific TNF mutant. Administration of the TNF mutant binding only to the p75 receptor did not induce any of these responses. We conclude that TNF-Induced stimulation of coagulation, fibrinolysis, neutrophil degranulation, and release of secretory phospholipase A2 are predominantly mediated by the p55 TNF receptor.

Animals↗
PubMed1996

CRP-mediated activation of complement in vivo: assessment by measuring circulating complement-C-reactive protein complexes.

The in vivo function of C-reactive protein (CRP) is unknown. Among the in vitro functions assigned to CRP is the ability to activate complement via the classical pathway. To date, there is no evidence supporting that CRP exerts this function in vivo. We here show a novel approach to assess CRP-mediated complement activation in vivo, which is based on the property that activated complement factors C3 and C4 fix to CRP during complement activation induced by this acute phase protein. We developed specific ELISAs for complexes between CRP and C4b, C4d, C3b, or C3d. We established that in vitro complement-CRP complexes were formed only during CRP-dependent activation, and not during activation by other activators, even in the presence of high CRP levels. Circulating levels of complement-CRP complexes were undetectable in normal donors, but significantly increased in nine patients following implantation of a renal allograft. Importantly, levels of complement-CRP complexes did not change in these patients upon a bolus infusion of mAb OKT3, which induces activation of the classical complement pathway, demonstrating in vivo that complement-CRP complexes are not formed during CRP-independent activation of complement, even when CRP is elevated. We conclude that measurement of complement-CRP complexes provides a suitable tool to study CRP-mediated activation of complement in vivo. Furthermore, increased levels of these complexes occur in clinical samples, indicating that CRP may induce activation of complement in vivo.

Adult↗
PubMed1996

Dissection of CR1, factor H, membrane cofactor protein, and factor B binding and functional sites in the third complement component.

Previous studies have suggested that the residues 727-768 of human (Hu) C3 contain the binding sites for CR1, factor H, and factor B. Here, we have (1) characterized further some of the C3 structural requirements for its binding to CR1, H, and B, (2) investigated the functions associated with these C3-ligand interactions, and (3) studied the relationship of MCP-binding sites in C3 with those for CR1, H, and B. Hu C3 molecules in which residues 727-768 were deleted (designated C3delta727-768) or substituted with the corresponding segment of cobra venom factor, Xenopus, or trout C3 (chimeric C3s) were expressed in the baculovirus system and analyzed for their reactivity with C3-binding proteins. In contrast to wild-type iC3 which, in the presence of CR1, is cleaved by factor I to iC3b-a and C3c-a and C3dg, all chimeric C3s were cleaved only to iC3b-a. In addition, the cleavage of deleted (C3delta727-768) iC3 to iC3b-a by factor I in the presence of CR1 was significantly reduced, whereas it remained unaltered in the presence of MCP. Cleavage of iC3 to iC3b-a by factor I and H was similar in all expressed C3s except C3delta727-768, whose cleavage was significantly reduced. All of the expressed molecules except C3delta727-768 were capable of forming the fluid-phase alternative pathway C3 convertase, and all reacted with properdin. These results suggest that during cleavage of iC3 by factor I and CR1, or H, CR1 and H bind to at least two sites on C3 and that the MCP binding site(s) on C3b are different from those for CR1. They also indicate that some or all of the C3 residues that are directly involved in, or contribute to, the structure of one of the CR1 and H binding sites are located within residues 727-768. These studies also demonstrate that, although this segment of C3 may be involved in C3-factor B interaction, other residues in addition to 736EE (previously implicated in B binding) must also contribute significantly to this interaction.

Amino Acid Sequence↗
PubMed1996

Release of interleukin-12 in experimental Escherichia coli septic shock in baboons: relation to plasma levels of interleukin-10 and interferon-gamma.

Interleukin (IL)-12 is thought to be a key factor for the induction of interferon gamma (IFN-gamma), a cytokine essential for the lethal effects of endotoxin. We report here on the release of the nonfunctional subunit of IL-12, p40, as well as biologically active heterodimeric IL-12, p70, after administration of a lethal (n = 5) or sublethal (n = 8) dose of live Escherichia coli to baboons. Remarkably, on lethal challenge, peak levels of p40 were observed at 3 hours that were about twofold lower than those elicited after sublethal challenge (2,813 +/- 515 pg/mL v 4,972 +/- 732 pg/mL, P < .05). This disparity was also observed, although to a lesser extent, for IL-12 p70 antigen, of which maximum levels of 91 +/- 47 pg/mL and 151 +/- 41 pg/mL were measured 6 hours after a lethal or sublethal dose of E coli, respectively. Circulating p70 antigen correlated with IL-12 biologic activity (r = 0.869; P < .001). When comparing lethal to sublethal conditions, lower peak levels of IL-12 on lethal E coli sharply contrasted with higher levels of other proinflammatory cytokines, such as tumor necrosis factor (TNF)-alpha, IL-1beta, IL-6, and IL-8 observed in these animals. Lower IL-12 concentrations in the lethal group may have resulted in part from the enhanced production of IL-10, a known inhibitor of IL-12 synthesis in vitro, as peak levels of this cytokine 3 hours postchallenge inversely correlated with peak levels of IL-12, in particular p40 (r = -0.802; P < .01). Contrary to what might be expected if IFN-gamma were solely induced by IL-12, lethally challenged baboons generated threefold more IFN-gamma at 6 hours than those receiving a sublethal dose (P < .05). Moreover, higher levels of IFN-gamma were associated with lower p40/p70 ratios, suggesting that, in agreement with observations in vitro, IFN-gamma may have preferentially upregulated the release of p70 over p40. These data show that IL-12 is released in experimental septic shock in nonhuman primates and suggest that IL-10 and IFN-gamma are involved in the regulation of this release. Furthermore, this study indicates that the systemic release of IL-12 might be essential, but is not likely sufficient, to promote lethal production of IFN-gamma in sepsis.

Animals↗
PubMed1996

Release of leukemia inhibitory factor in primate sepsis. Analysis of the role of TNF-alpha.

Leukemia inhibitory factor (LIF), a pleiotropic cytokine with many biologic effects overlapping with those of IL-6, has been implicated in the pathogenesis of sepsis. We here analyzed the kinetics of LIF in 13 baboons challenged with a lethal (n=6) or sublethal (n=7) dose of Escherichia coli. In addition, to assess the role of TNF-alpha in the induction of LIF in vivo, seven baboons were studied that had either received a bolus injection of recombinant human TNF-alpha (100 micrograms/kg, n=3), or to whom 15 mg/kg of an anti-TNF mAB before lethal E. coli challenge was administered (n=4). LIF levels increased 2 h after E coli challenge, and reached maximum values at 4 and 8 h after a sublethal (4.4 +/- 1.6 ng/ml) or lethal (40.9 +/- 3.8 ng/ml) dose, respectively. TNF-alpha injection induced a modest rise in LIF concentrations, peaking after 6 h (228 +/- 46 pg/ml). Circulating LIF correlated with plasma levels of IL-6, both after E. coli challenge (Spearman Rank coefficient of correlation (r) = 0.849, p<0.001), as well as upon TNF-alpha injection (r=0.863, p<0.001). Moreover, the E. coli-induced release of either cytokine was reduced 6- to 10-fold after pretreatment with anti-TNF mAb, except in one nonsurviving animal, which exhibited a progressive increase of LIF and IL-6 levels despite the absence of TNF immunoreactivity. These results show that TNF-alpha is an intermediate factor in concerted release of LIF and IL-6 in vivo, and indicate that the enhanced elaboration of these cytokines may predict disease outcome in severe sepsis.

Animals↗
PubMed1996

Modulation of contact system proteases by glycosaminoglycans. Selective enhancement of the inhibition of factor XIa.

We investigated the influence of dextran sulfate, heparin, heparan sulfate, and dermatan sulfate on the inhibition of FXIa (where FXIa is activated factor XI, for example), FXIIa, and kallikrein by C1 inhibitor, alpha1-antitrypsin, alpha2-antiplasmin, and antithrombin III. The second-order rate constants for the inhibition of FXIa by C1 inhibitor, alpha1-antitrypsin, alpha2-antiplasmin, and antithrombin III, in the absence of glycosaminoglycans, were 1.8, 0.1, 0.43, and 0.32 x 10(3) M-1 s-1, respectively. The rate constants of the inactivation of FXIa by C1 inhibitor and by antithrombin III increased up to 117-fold in the presence of glycosaminoglycans. These data predicted that considering the plasma concentration of the inhibitors, C1 inhibitor would be the main inhibitor of FXIa in plasma in the presence of glycosaminoglycans. Results of experiments in which the formation of complexes between serine protease inhibitors and FXIa was studied in plasma agreed with this prediction. Glycosaminoglycans did not enhance the inhibition of alpha-FXIIa, beta-FXIIa, or kallikrein by C1 inhibitor. Thus, physiological glycosaminoglycans selectively enhance inhibition of FXIa without affecting the activity of FXIIa and kallikrein, suggesting that glycosaminoglycans may modulate the biological effects of contact activation, by inhibiting intrinsic coagulation without affecting the fibrinolytic potential of FXIIa/kallikrein.

Amides↗
PubMed1996

Structure/function analysis of human factor XII using recombinant deletion mutants. Evidence for an additional region involved in the binding to negatively charged surfaces.

The binding site of human factor XII (FXII) for negatively charged surfaces has been proposed to be localized in the N-terminal region of factor XII. We have generated two recombinant factor XII proteins that lack this region: one protein consisting of the second growth-factor-like domain, the kringle domain, the proline-rich region and the catalytic domain of FXII (rFXII-U-like), and another consisting of only 16 amino acids of the proline-rich region of the heavy-chain region and the catalytic domain (rFXII-1pc). Each recombinant truncated protein, as well as recombinant full-length FXII (rFXII), were produced in HepG2 cells and purified by immunoaffinity chromatography. The capability of these recombinant proteins to bind to negatively charged surfaces and to initiate contact activation was studied. Radiolabeled rFXII-U-like and, to a lesser extent, rFXII-lpc bound to glass in a concentration-dependent manner, yet with lower efficiency than rFXII. The binding of the recombinant proteins was inhibited by a 100-fold molar excess of non-labeled native factor XII. On native polyacrylamide gel electrophoresis, both truncated proteins appeared to bind also to dextran sulfate, a soluble negatively charged compound. Glass-bound rFXII-U-like was able to activate prekallikrein in FXII-deficient plasma (assessed by measuring the generation of kallikrein-C1-inhibitor complexes), but less efficiently than rFXII, rFXII-U-like and rFXII-lpc exhibited coagulant activity, but this activity was significantly lower than that of rFXII. These data confirm that the N-terminal part of the heavy-chain region of factor XII contains a binding site for negatively charged activating surfaces, and indicate that other sequences, possibly located on the second epidermal-growth-factor-like domain and/or the kringle domain, contribute to the binding of factor XII to these surfaces.

Base Sequence↗
PubMed1996

Expression of human recombinant granzyme A zymogen and its activation by the cysteine proteinase cathepsin C.

Human granzyme A is one of the serine proteinases present in the granules of cytotoxic T lymphocytes and natural killer cells. Granzymes are synthesized as inactive proenzymes with an amino-terminal prodipeptide, which is processed during transport of granzymes to the cytotoxic granules, where they are stored as active proteinases. In this study, we explored the possibility of producing recombinant granzymes. Recombinant human granzyme A zymogen was expressed in several eukaryotic cell lines (HepG2, Jurkat, and COS-1) after infection with a recombinant vaccinia virus containing full-length granzyme A cDNA. Immunoblot analysis of cell lysates showed that all infected cells produced a disulfide-linked homodimer of identical molecular weight as natural granzyme A. Infected HepG2 cells produced the largest amount of this protease (approximately 160 times more than lymphokine activated killer (LAK) cells). The recombinant protein only had high mannose type oligosaccharides as did the natural protein. Although infected HepG2 and COS cells contained high granzyme A antigen levels, lysates from these cells did not show any granzyme A proteolytic activity. However, the inactive proenzyme could be converted into active granzyme A by incubation with the thiol proteinase cathepsin C (dipeptidyl peptidase I). This study is the first to demonstrate expression of an active recombinant human cytotoxic lymphocyte proteinase and conversion of inactive progranzyme A into an active enzyme by cathepsin C. We suggest that a similar approach can be used for the production of other granzymes and related proteinases.

Animals↗
PubMed1996

Inhibition of factor XII in septic baboons attenuates the activation of complement and fibrinolytic systems and reduces the release of interleukin-6 and neutrophil elastase.

In previous studies, we have shown that administration of monoclonal antibody (MoAb) C6B7 against human factor XII to baboons challenged with a lethal dose of Escherichia coli abrogates activation of the contact system and modulates secondary hypotension. To evaluate the contribution of activated contact proteases to the appearance of other inflammatory mediators in this experimental model of sepsis, we studied the effect of administration of MoAb C6B7 on activation of complement and fibrinolytic cascades, stimulation of neutrophil degranulation, and release of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Activation of the complement system, as reflected by circulating C3b/c and C4b/c levels, was significantly reduced in five animals that had received MoAb C6B7 before a lethal dose of E coli as compared with five control animals that had been given a lethal challenge only. Inhibition of contact activation also modulated the fibrinolytic response, since the release of tissue-type plasminogen activator (t-PA) and the appearance of plasmin-alpha2-antiplasmin (PAP) complexes into the circulation was significantly attenuated upon pretreatment with anti-factor XII MoAb. In contrast, plasma levels of plasminogen activator inhibitor (PAI) were modestly enhanced in the treatment group. Degranulation of neutrophils, as assessed by circulating elastase-alpha1-protease inhibitor complexes, and release of IL-6 but not of TNF-alpha was decreased in anti-factor XII-treated animals. Observed differences in the inflammatory response between treatment and control groups were not likely due to different challenges, since the number of E coli that had been infused, as well as circulating levels of endotoxin after the challenge, were similar for both groups. These data suggest that activation of the contact system modulates directly or indirectly various mediator systems involved in the inflammatory response during severe sepsis in nonhuman primates.

Animals↗