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Biomedical subjects

C Erik Hack

Publications and source records attributed to C Erik Hack.

At least 73 records · Page 4Linked to original sources
PubMed2003

Functional characterization of the lectin pathway of complement in human serum.

Mannan-binding lectin (MBL) is a major initiator of the lectin pathway (LP) of complement. Polymorphisms in exon 1 of the MBL gene are associated with impaired MBL function and infections. Functional assays to assess the activity of the classical pathway (CP) and the alternative pathway (AP) of complement in serum are broadly used in patient diagnostics. We have now developed a functional LP assay that enables the specific quantification of autologous MBL-dependent complement activation in human serum. Complement activation was assessed by ELISA using coated mannan to assess the LP and coated IgM to assess the CP. Normal human serum (NHS) contains IgG, IgA and IgM antibodies against mannan, as shown by ELISA. These antibodies are likely to induce CP activation. Using C1q-blocking and MBL-blocking mAb, it was confirmed that both the LP and the CP contribute to complement activation by mannan. In order to quantify LP activity without interference of the CP, LP activity was measured in serum in the presence of C1q-blocking Ab. Activation of serum on coated IgM via the CP resulted in a dose-dependent deposition of C1q, C4, C3, and C5b-9. This activation and subsequent complement deposition was completely inhibited by the C1q-blocking mAb 2204 and by polyclonal Fab anti-C1q Ab. Evaluation of the LP in the presence of mAb 2204 showed a strong dose-dependent deposition of C4, C3, and C5b-9 using serum from MBL-wildtype (AA) but not MBL-mutant donors (AB or BB genotype), indicating that complement activation under these conditions is MBL-dependent and C1q-independent. Donors with different MBL genotypes were identified using a newly developed oligonucleotide ligation assay (OLA) for detection of MBL exon 1 polymorphisms. We describe a novel functional assay that enables quantification of autologous complement activation via the LP in full human serum up to the formation of the membrane attack complex. This assay offers novel possibilities for patient diagnostics as well as for the study of disease association with the LP.

Antibodies↗
PubMed2003

Rat C-reactive protein activates the autologous complement system.

Activation of complement is a biological function of human C-reactive protein (hCRP), whereas rat CRP (rCRP) has been claimed to be unable to activate complement. As important biological functions of proteins are probably conserved among species, we re-evaluated, using various ligands, the capability of rCRP to activate complement. The activation of complement by hCRP and rCRP was investigated in solid- and fluid-phase systems. In the solid-phase system, purified CRP was fixed to enzyme-linked immunosorbent assay (ELISA) plates and incubated with human or rat recalcified plasma. Dose-dependent binding of human and rat C3 and C4 was observed to human and rat CRP, respectively. In the fluid-phase system, recalcified rat plasma, which contains about 500 mg/l of CRP, or human plasma supplemented with hCRP, were incubated with lyso-phosphatidylcholine. A dose-dependent activation of complement was observed upon incubation with this ligand, as reflected by the generation of activated C4 as well as of CRP-complement complexes. This activation was, in both cases, inhibited by preincubation of plasma with p-aminophosphorylcholine, a specific inhibitor of the interaction of CRP with its ligands, or by chelation of calcium ions. We conclude that rat CRP, similarly to human CRP, can activate autologous complement. These results support the notion that opsonization of ligands with complement is an important biological function of CRP.

Animals↗
PubMed2003

Activation of protein C following infusion of protein C concentrate in children with severe meningococcal sepsis and purpura fulminans: a randomized, double-blinded, placebo-controlled, dose-finding study.

BACKGROUND: Meningococcal septic shock in children results in high mortality and morbidity, and decreased protein C levels in these patients are associated with a poor outcome. We carried out a randomized, double-blinded, placebo-controlled study by supplying protein C concentrate. This phase 2 study was designed to assess the activation process of protein C and to study the dosing regimen of protein C concentrate in children with purpura fulminans and meningococcal septic shock in the perspective of a possible phase 3 trial. METHODS: Forty children were randomized to receive placebo or protein C concentrate (200 IU/kg, 400 IU/kg, or 600 IU/kg), for a maximum of 7 days. Clinical and laboratory data, including plasma levels of protein C and activated protein C (APC), were collected at various time points. All patients received standard therapy for septic shock, including antibiotics, inotropic/vasoactive drugs, and blood products. RESULTS: Increased APC levels relative to baseline were observed for the 27 of 28 patients treated with protein C concentrate, and the areas under the curve of protein C and APC were correlated with the dosage of protein C concentrate administered. Activation of coagulation, as evidenced by d-dimer levels, as well as the ratio of thrombin vs. APC normalized significantly faster with increasing dosages of protein C concentrate. No adverse reactions related to protein C concentrate were observed. Nine of the 40 (23%) patients died, and five survivors required amputations, with no differences in these rates among the randomized groups. Baseline APC levels were positively correlated with sequential organ failure assessment and pediatric risk of mortality scores and with d-dimers, tumor necrosis factor-alpha, interleukin-1, interleukin-6, interleukin-8, plasminogen activator inhibitor-1, TAT complexes, and PAP complexes. CONCLUSIONS: Treatment with protein C concentrate is safe in children with purpura fulminans and meningococcal septic shock and leads to dose-related increases of plasma APC and resolution of coagulation imbalances.

Anticoagulants↗
PubMed2003

Elevated nucleosome levels in systemic inflammation and sepsis.

OBJECTIVE: Multiple organ dysfunction syndrome is a frequent complication of severe sepsis and septic shock and has a high mortality. We hypothesized that extensive apoptosis of cells might constitute the cellular basis for this complication. DESIGN: Retrospective study. SETTING: Medical and surgical wards or intensive care units of two university hospitals. PATIENTS: Fourteen patients with fever, 15 with systemic inflammatory response syndrome, 32 with severe sepsis, and eight with septic shock. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We assessed circulating levels of nucleosomes, specific markers released by cells during the later stages of apoptosis, with a previously described enzyme-linked immunosorbent assay in these 69 patients with fever, systemic inflammatory response syndrome, severe sepsis, or septic shock. Severity of multiple organ dysfunction syndrome was assessed with sepsis scores, and clinical and laboratory variables. Elevated nucleosome levels were found in 64%, 60%, 94%, and 100% of patients with fever, systemic inflammatory response syndrome, severe sepsis, or septic shock, respectively. These levels were significantly higher in patients with septic shock as compared with patients with severe sepsis, systemic inflammatory response syndrome, or fever, and in nonsurvivors as compared with survivors. In patients with advanced multiple organ dysfunction syndrome, nucleosome levels correlated with cytokine plasma levels as well as with variables predictive for outcome. CONCLUSIONS: Patients with severe sepsis and septic shock have elevated plasma levels of nucleosomes. We suggest that apoptosis, probably resulting from exposure of cells to excessive amounts of inflammatory mediators, might by involved in the pathogenesis of multiple organ dysfunction syndrome.

Adult↗
PubMed2003

Administration of C1 inhibitor reduces neutrophil activation in patients with sepsis.

Forty patients with severe sepsis or septic shock recently received C1 inhibitor. In the present study we studied the effect of C1 inhibitor therapy on circulating elastase-alpha(1)-antitrypsin complex (EA) and lactoferrin (LF) levels in these patients to gain further insight about agonists involved in the activation of neutrophils in human sepsis. Elevated levels of EA and LF were found in 65 and 85% of the septic patients, respectively. Patients with elevated EA levels had higher organ dysfunction scores, higher levels of cytokines, and higher levels of complement activation products than patients with normal EA levels. C1 inhibitor therapy reduced EA as well as complement activation and IL-8 release in the patients with elevated EA on admission. We conclude that neutrophil activation in human sepsis correlates with the severity of organ dysfunction and involves complement and interleukin-8 as agonists. The effect of C1 inhibitor therapy on neutrophils may provide an explanation for the beneficial, although mild, effects of this treatment on organ dysfunction in sepsis.

Adult↗
PubMed2002

Effects of IC14, an anti-CD14 antibody, on coagulation and fibrinolysis during low-grade endotoxemia in humans.

To determine the role of CD14 in lipopolysaccharide (LPS)-induced effects on coagulation and fibrinolysis in humans, 16 healthy subjects received an intravenous injection of LPS preceded by intravenous IC14, a recombinant chimeric monoclonal antibody against human CD14, or placebo. LPS-induced coagulation activation (tissue-factor mRNA in whole blood cells and plasma concentrations of F1+2) was not influenced by IC14, whereas the antibody reduced the increase in thrombin-antithrombin complexes and soluble fibrin. LPS injection also was associated with an early activation of fibrinolysis (plasma concentrations of tissue-type plasminogen activator and plasmin-alpha(2)-antiplasmin complexes), followed by an inhibitory response (plasminogen activator inhibitor type 1), which were attenuated by IC14. Furthermore, LPS reduced thrombin-activatable fibrinolysis-inhibitor antigen levels and increased soluble thrombomodulin levels, which were not influenced by IC14. These results suggest that different hemostatic responses during endotoxemia may proceed via CD14-dependent and -independent pathways.

Adult↗
PubMed2002

Expression levels of apoptosis-related proteins predict clinical outcome in anaplastic large cell lymphoma.

In vitro studies suggest that resistance to chemotherapy-induced apoptosis might explain poor response to therapy in fatal cases. Actual execution of apoptosis depends on proper functioning of effector caspases, particularly caspase 3, and on the expression levels of apoptosis-regulating proteins, including Bcl-2 and the recently identified granzyme B- specific protease inhibitor 9 (PI9). Thus, high levels of caspase 3 activation should reflect proper functioning of the apoptosis pathways, resulting in chemotherapy-sensitive neoplastic cells and a favorable prognosis. We tested this hypothesis by quantifying numbers of tumor cells positive for active caspase 3, Bcl-2, and PI9, respectively, in pretreatment biopsies of systemic anaplastic large cell lymphoma (ALCL) patients and by comparing these numbers with clinical outcome. Activation of caspase 3 in more than 5% of the tumor cells was strongly correlated with a highly favorable outcome. High numbers of Bcl-2- and PI9-positive tumor cells were found to predict unfavorable prognosis. This prognostic effect was strongly related to anaplastic lymphoma kinase (ALK) status: ALK-positive ALCL had significantly higher levels of active caspase 3, while high expression of the antiapoptotic proteins Bcl-2 and PI9 was almost completely restricted to ALK-negative cases. In conclusion, high numbers of active caspase 3-positive tumor cells predict a highly favorable prognosis in systemic ALCL patients. Poor prognosis is strongly related to high numbers of Bcl-2- and PI9-positive neoplastic cells. These data support the notion that a favorable response to chemotherapy depends on an intact apoptosis cascade. Moreover, these data indicate that differences in prognosis between ALK-positive and ALK-negative ALCL might be explained by differences in expression of apoptosis-inhibiting proteins.

Adult↗
PubMed2002

Anti-inflammatory effects of a p38 mitogen-activated protein kinase inhibitor during human endotoxemia.

The p38 mitogen-activated protein kinase (MAPK) participates in intracellular signaling cascades resulting in inflammatory responses. Therefore, inhibition of the p38 MAPK pathway may form the basis of a new strategy for treatment of inflammatory diseases. However, p38 MAPK activation during systemic inflammation in humans has not yet been shown, and its functional significance in vivo remains unclear. Hence, we exposed 24 healthy male subjects to an i.v. dose of LPS (4 ng/kg), preceded 3 h earlier by orally administered 600 or 50 mg BIRB 796 BS (an in vitro p38 MAPK inhibitor) or placebo. Both doses of BIRB 796 BS significantly inhibited LPS-induced p38 MAPK activation in the leukocyte fraction of the volunteers. Cytokine production (TNF-alpha, IL-6, IL-10, and IL-1R antagonist) was strongly inhibited by both low and high dose p38 MAPK inhibitor. In addition, p38 MAPK inhibition diminished leukocyte responses, including neutrophilia, release of elastase-alpha(1)-antitrypsin complexes, and up-regulation of CD11b with down-regulation of L-selectin. Finally, blocking p38 MAPK decreased C-reactive protein release. These data identify p38 MAPK as a principal mediator of the inflammatory response to LPS in humans. Furthermore, the anti-inflammatory potential of an oral p38 MAPK inhibitor in humans in vivo suggests that p38 MAPK inhibitors may provide a new therapeutic option in the treatment of inflammatory diseases.

Administration, Oral↗
PubMed2002

Novel strategy for the selection of human recombinant Fab fragments to membrane proteins from a phage-display library.

Traditionally, the selection of phage-display libraries is performed on purified antigens (Ags), immobilized to a solid substrate. However, this approach may not be applicable for some Ags, such as membrane proteins, which for structural integrity strongly rely on their native environment. Here we describe an approach for the selection of phage-libraries against membrane proteins. The envelope glycoproteins (Env) of the Human Immunodeficiency Virus type-1 (HIV-1) were used as a model for a type-1 integral membrane protein. HIV-1IHI Env, expressed on the surface of Rabbit Kidney cells (RK13) with a recombinant vaccinia virus (rVV), was solubilized using the non-ionic detergent n-Octyl beta-D-glucopyranoside (OG). Membrane associated Env was reconstituted into vesicles by the simultaneous removal of detergent and free monomeric Env subunits by gel-filtration. The resulting antigen preparation, termed OG-P1IHI, was captured on microtiter plates coated with Galanthus nivalis agglutinin (GNA) and used for rounds of selection (panning) of a well-characterized phage-display library derived from an HIV-1 seropositive donor. Simultaneously, an identical experiment was performed with OG-P1IHI vesicles disrupted by Nonidet P-40 (NP-P1IHI). Both membrane-associated and soluble Ags were selected for vaccinia-specific clones (OG-P1IHI: 59/75 and NP-P1IHI: 1/75) and HIV-1-specific clones (OG-P1IHI: 11/75 and NP-P1IHI: 65/75) using our approach. Hence, the novel panning strategy described here may be applicable for selection of phage-libraries against membrane proteins.

Amino Acid Sequence↗
PubMed2002

Expression of the granzyme B inhibitor, protease inhibitor 9, by tumor cells in patients with non-Hodgkin and Hodgkin lymphoma: a novel protective mechanism for tumor cells to circumvent the immune system?

In tumor cells, the serine protease granzyme B is the primary mediator of apoptosis induced by cytotoxic T lymphocytes (CTLs)/natural killer (NK) cells. The human intracellular serpin proteinase inhibitor 9 (PI9) is the only known human protein able to inhibit the proteolytic activity of granzyme B. When present in the cytoplasm of T lymphocytes, PI9 is thought to protect CTLs against apoptosis induced by their own misdirected granzyme B. Based on the speculation that tumors may also express PI9 to escape CTL/NK cell surveillance, immunohistochemical studies on the expression of PI9 in various lymphomas were performed. Ninety-two cases of T-cell non-Hodgkin lymphoma (NHL), 75 cases of B-cell NHL, and 57 cases of Hodgkin lymphomas were stained with a PI9-specific monoclonal antibody. In T-cell NHL, highest PI9 expression was found in the extranodal T-cell NHL. In nearly 90% of enteropathy-type T-cell NHLs and 80% of NK/T-cell, nasal-type lymphomas, the majority of the tumor cells expressed PI9. In nodal T-anaplastic large cell lymphomas and peripheral T-cell lymphomas (not otherwise specified), PI9 expression occurred less frequently. In B-cell NHL, PI9 expression was associated with high-grade malignancy; 43% of diffuse large B-cell lymphomas showed PI9(+) tumor cells. Finally, PI9 expression was also found in 10% of Hodgkin lymphomas. This is the first report describing the expression of the granzyme B inhibitor PI9 in human neoplastic cells in vivo. Expression of this inhibitor is yet another mechanism used by tumor cells to escape their elimination by cytotoxic lymphocytes.

Antibodies, Monoclonal↗
PubMed2002

Cell-derived microparticles in synovial fluid from inflamed arthritic joints support coagulation exclusively via a factor VII-dependent mechanism.

OBJECTIVE: To determine the cellular origin of synovial microparticles, their procoagulant properties, and their relationship to local hypercoagulation. METHODS: Microparticles in synovial fluid and plasma from patients with rheumatoid arthritis (RA; n = 10) and patients with other forms of arthritis (non-RA; n = 10) and in plasma from healthy subjects (n = 20) were isolated by centrifugation. Microparticles were identified by flow cytometry. The ability of microparticles to support coagulation was determined in normal plasma. Concentrations of prothrombin fragment F(1+2) (by enzyme-linked immunosorbent assay [ELISA]) and thrombin-antithrombin (TAT) complexes (by ELISA) were determined as estimates of the coagulation activation status in vivo. RESULTS: Plasma from patients and healthy controls contained comparable numbers of microparticles, which originated from platelets and erythrocytes. Synovial microparticles from RA patients and non-RA patients originated mainly from monocytes and granulocytes; few originated from platelets and erythrocytes. Synovial microparticles bound less annexin V (which binds to negatively charged phospholipids) than did plasma microparticles, exposed tissue factor, and supported thrombin generation via factor VII. F(1+2) (median 66 nM) and TAT complex (median 710 microg/liter) concentrations were elevated in synovial fluid compared with plasma from the patients (1.6 nM and 7.0 microg/liter, respectively) as well as the controls (1.0 nM and 2.9 microg/liter, respectively). CONCLUSION: Synovial fluid contains high numbers of microparticles derived from leukocytes that are strongly coagulant via the factor VII-dependent pathway. We propose that these microparticles contribute to the local hypercoagulation and fibrin deposition in inflamed joints of patients with RA and other arthritic disorders.

Adult↗
PubMed2002

Impaired fibrinolysis in the pathogenesis of dengue hemorrhagic fever.

The mechanisms contributing to bleeding complications in dengue hemorrhagic fever were studied by investigating the pattern of activation of the coagulation and fibrinolytic systems in 50 children with severe dengue hemorrhagic fever. Thirteen patients (26%) died, and activation of coagulation was most pronounced in the deceased group. Fibrinolysis was also activated, but this activation was relatively weak compared with that of coagulation as a result of persistently high plasminogen activator inhibitor levels. Plasminogen activator inhibitor also prevented a switch from the procoagulant to the profibrinolytic state in lethal dengue hemorrhagic fever, which was further enhanced by an acquired protein C deficiency. The present study is the first to demonstrate such a mechanism in a viral infection. This imbalance between coagulation and fibrinolysis may be used as a prognostic marker, but it may also be a target for future therapeutic intervention.

Biomarkers↗
PubMed2002

Secretory type II phospholipase A(2) binds to ischemic myocardium during myocardial infarction in humans.

OBJECTIVE: An increase of circulating secretory Phospholipase A(2) (sPLA(2)) is a risk factor for coronary artery disease. We hypothesized that this reflects participation of sPLA(2) in local inflammatory reactions ensuing in ischemic myocardium. Therefore, we studied the course of circulating sPLA(2), in patients with acute myocardial infarction (AMI) or unstable angina pectoris (UAP), and investigated the presence of sPLA(2) in infarcted myocardial tissue. METHODS: Plasma samples of 107 patients with AMI or UAP, collected on admission and at varying intervals thereafter, were tested for the presence of sPLA(2) and C-reactive protein (CRP). Cumulative release values of these parameters were calculated, which allowed for comparison of the results rearranged in time according to the onset of symptoms. By immunohistochemistry we studied the presence of sPLA(2) and CRP in myocardial tissue of 30 patients who died subsequent to AMI. RESULTS: Levels of sPLA(2) became elevated during the disease course in 66 of the 87 patients with AMI, and were higher than those of the patients with UAP of whom 8 of the 20 had elevated levels. By immunohistochemistry sPLA(2) was found to be localized in the infarcted myocardium, particularly in its borderzone, from 12 h after the onset of AMI. Positive staining for sPLA(2) was more extensive than that for CRP. CONCLUSIONS: The localization pattern of sPLA(2) in infarcted myocardium as well as its plasma course, in relation to those of CRP, are in line with a supposed pro-inflammatory role during AMI for sPLA(2) as a generator of lysophospholipids serving as ligands for CRP.

Angina, Unstable↗
PubMed2002

Granzyme expression in fine-needle aspirates from liver allografts is increased during acute rejection.

We investigated whether determination in fine-needle aspiration biopsy (FNAB) specimens of cells expressing granzymes (Grs) and Fas ligand would provide a reliable, easy, and quantitative measure of rejection activity in the transplanted liver. Retrospectively, 13 FNAB specimens obtained during clinical acute rejection, 10 FNAB specimens obtained during subclinical rejection, 12 FNAB specimens obtained during cytomegalovirus (CMV) infection, and 26 FNAB specimens obtained in the absence of rejection or infection were included on the study. Cytospin preparations of FNAB and peripheral-blood specimens were immunocytochemically stained for Fas-ligand and Gr, and increments in the liver were calculated by subtracting frequencies of positive cells in blood from those in FNAB specimens. Only sporadically Fas ligand-expressing, but many Gr-expressing, cells were detected in FNAB specimens. Increments in Gr-positive (Gr(+)) cells were significantly greater in FNAB specimens obtained during clinical rejection (median, 70 Gr(+) cells; range, 0 to 312 Gr(+) cells; P = .006) and tended to be greater in FNAB specimens obtained during subclinical rejection (median, 62 Gr(+) cells; range, 5 to 113 Gr(+) cells; P = .09) compared with those obtained in the absence of rejection (median, 16 Gr(+) cells; range, 0 to 103 Gr(+) cells). Increments obtained during clinical or subclinical rejection did not differ from those obtained during CMV infection (median, 27 Gr(+) cells; range, 6 to 212 Gr(+) cells). With the exclusion of specimens obtained during CMV infection, the sensitivity of Gr determination in FNAB specimens for the diagnosis of acute rejection (either clinical or subclinical) was 70%, and specificity, 69%. In FNAB specimens obtained during clinical and subclinical acute rejection episodes after liver transplantation, increased numbers of Gr-expressing cells were present; in the absence of CMV infection, their quantification provides a measure for rejection activity with moderate accuracy.

Biopsy, Needle↗
PubMed2002

Role of complement in graft rejection after organ transplantation.

Activation of the complement system may significantly contribute to the inflammatory reaction after solid organ transplantation. In allotransplantation, the complement system may be activated by ischemia/reperfusion and, possibly, by antibodies directed against the graft. In xenotransplantation from nonprimates to primates, the major activators for complement are preexisting antibodies. Studies in animal models have shown that the use of complement inhibitors may significantly prolong graft survival. This review describes the role of the complement system in organ injury after organ transplantation and the use of complement inhibitors to prevent damage to the graft after allo- or xenotransplantation.

Animals↗