Biomedical subjects

Lucien A Aarden

Publications and source records attributed to Lucien A Aarden.

8 recordsLinked to original sources

PubMed2006

Mannan-binding lectin (MBL)-mediated opsonization is enhanced by the alternative pathway amplification loop.

The complement system is a humoral effector in the innate immune system. Three activation pathways exist in the complement system, known as the classical pathway, the lectin pathway and the alternative pathway. Dysfunction of lectin pathway activation is caused by MBL deficiency. MBL deficiency in a cohort of healthy Caucasian blood bank donors was investigated with MBL genotyping and MBL plasma concentration. Recognition of the yeast-derived zymosan by MBL was investigated with Western blot. The involvement of the alternative pathway amplification loop in enhancing MBL-mediated opsonization of zymosan was investigated in a novel opsonophagocytosis assay for flow cytometry. Sera deficient for MBL, factor D or properdin were tested, and purified MBL, factor D or properdin were used to recover opsonization. The optimal receiver-operator characteristic (ROC) cut-off value for dividing the Caucasian cohort in MBL-sufficient and MBL-deficient was calculated at 0.7 microg/ml. Thirty-eight percent of the group had concentrations below 0.7 microg/ml. Zymosan eluates opsonized with MBL-sufficient sera contain high oligomers of MBL, while eluates from MBL-deficient donors contained hardly any MBL. The MBL-, factor D- and properdin-deficient sera showed reduced opsonophagocytosis by human control neutrophils, as compared to normal MBL-sufficient sera. This reduction in opsonization was restored to normal levels by addition of purified MBL, factor D and properdin. The absence of opsonization in the factor D- and properdin-deficient sera, but presence in normal serum after blocking with anti-C1q-F(ab)2 and anti-MBL-F(ab)2, demonstrates the involvement of the amplification loop in MBL-initiated zymosan opsonization, even at very low serum concentrations (up to 3%, v/v). In conclusion, our data demonstrate that the MBL-mediated route of complement activation depends on the alternative pathway amplification loop for optimal opsonization of zymosan.

Cohort Studies↗

PubMed2006

Variable mannose-binding lectin expression during postoperative acute-phase response.

BACKGROUND: Low plasma concentrations and genetic polymorphisms of mannan-binding lectin (MBL) have been associated with infectious disease complications during various conditions. The present study examined the nature and expression of MBL deficiency during a surgery-induced acute-phase response. METHODS: Blood was sampled from 20 consecutive patients before and 1, 3, 5, 7, and 10 days and 6 weeks after a uniform abdominal operation (transhiatal esophagectomy). Plasma concentrations of MBL, C-reactive protein (CRP), and secretory phospholipase A2 (sPLA2) were measured. Patients were classified as low- or high-level MBL producers by their preoperative concentration (<0.5 or > or = 0.5 micrograms/mL), and were cross-verified for actual MBL deficiency by nucleotide sequencing of both the MBL promoter and exon-1 alleles. RESULTS: Baseline plasma MBL concentrations correlated with maximal postoperative plasma concentrations (r = 0.88; p < 0.0001). This was not found for CRP and sPLA2 (r = 0.19 and r = 0.08, respectively). Alleles responsible for structural MBL variants were detected in 40% of patients and were associated with significantly reduced MBL concentrations (p = 0.005). The baseline cut-off value in plasma of 0.5 micrograms/mL clearly identified individuals with variant exon-1 alleles (sensitivity 100%, specificity 83%). CONCLUSIONS: Baseline MBL plasma concentrations are predictive of MBL expression during the acute-phase response. A baseline cut-off value of 0.5 micrograms/mL can be used to identify patients with variants in the exon-1 region of the MBL gene without the need for nucleotide sequencing. Clinical studies may use this easy and quick method to identify MBL deficient patients preoperatively, as they are conditionally at risk for infectious complications.

Acute-Phase Reaction↗

PubMed2005

State and diagnostic value of plasma tissue factor in early-hospitalised patients with chest pain.

To study the state and diagnostic value of plasma tissue factor (TF) in patients with acute coronary syndromes (ACS), we quantitatively compared plasma TF antigen and TF activity in 90 early-hospitalised patients with chest pain. Using high-affinity antibodies, a sensitive assay for TF antigen was developed with a detection limit of 40 fmol/l. One of the antibodies was used to capture TF from plasma and, after elution and dialysis-free reconstitution in phospholipid-glucoside micelles, absolute amounts of TF activity could be measured with a detection limit of 80 fmol/l. All TF in plasma was found to be exposed, and a value of 2.5(1.1-14.8) pmol/l (median with range) was found for TF antigen. Most of this TF antigen (70-80%) circulated in a (potentially) functional state. Left in its in vivo state, however, TF captured from plasma was totally inactive, probably because of the lack of a procoagulant matrix. Compared with controls with non-cardiac chest pain, TF activity was unchanged and TF antigen about 25% elevated in ACS patients. Combined with the markers prothrombin fragment F1+2 and fatty acid-binding protein, TF did not improve the early diagnosis of ACS.

Acute Disease↗

PubMed2005

High-affinity antibodies in a new immunoassay for plasma tissue factor: reduction in apparent intra-individual variation.

Tissue factor, the main initiator of blood coagulation, is shed into plasma by blood cells and endothelium. While studying such circulating plasma tissue factor with a commercially available immunoassay, we found unsatisfactory results and therefore developed a new and highly sensitive enzyme-linked immunosorbent assay (ELISA). High-affinity monoclonal antibodies raised against recombinant soluble tissue factor were used and the new assay had a detection limit of 40 fmol/L, approximately six-fold lower than existing assays. Normal ranges in 20 healthy donors were established in serum and in citrated EDTA and heparinized plasma. Tissue factor was also measured in three successive plasma samples from 43 patients with type 2 diabetes mellitus. In citrated plasma from healthy donors, tissue factor concentrations were 2.5 (1.0-9.3) pmol/L (median with range) and were not significantly different in diabetics. With a commercially available immunoassay, seven plasma samples were below the detection limit. Use of the new assay reduced intra-individual variation in diabetics from 49% to 14% and we conclude that high-affinity antibodies may markedly improve immunoassay performance.

Adolescent↗

PubMed2005

4-Hydroxy-oxyphenbutazone is a potent inhibitor of cytokine production.

4-Hydroxy-oxyphenbutazone (4OH-OPB), is currently in phase II trials for its immunosuppressive effect in patients with rheumatoid arthritis. 4OH-OPB and other compounds related to phenylbutazone were tested for their effect on in vitro cytokine production by monocytes and lymphocytes present in peripheral mononuclear cells (PBMC) or whole blood (WB) cultures, and compared against phenylbutazone and oxyphenbutazone, two known anti-inflammatory drugs. In PBMC cultures, 4OH-OPB was by far the most potent inhibitor, and both monokines and Th1 and Th2 lymphokines were efficiently inhibited at low concentrations. In WB cultures, 4OH-OPB was less effective than in PBMC cultures, but was still the best inhibitor of lymphokine production and, furthermore, was the only inhibitor of monokine production. The increase in 4OH-OPB concentration needed to induce the same inhibition of cytokine production in WB as in PBMC culture could be mimicked by the addition of erythrocytes to the PBMC cultures. Experiments with radioactively-labeled 4OH-OPB suggest that 4OH-OPB is taken up very rapidly into erythrocytes and is secreted by the erythrocytes with much slower kinetics via a multidrug-resistance-associated protein. The secreted compound is most likely structurally different from 4OH-OPB, as in PBMC and WB cultures, the inhibition of cytokine production seems to be caused by a different mechanism. In PBMC cultures, the inhibition of cytokine production is accompanied by a loss of cell viability, while this is not the case when 4OH-OPB inhibits cytokine production in WB. Our data suggest that 4OH-OPB may be useful as an immunosuppressive drug for patients with inflammatory diseases.

Cell Survival↗

PubMed2004

Complement activation by apoptotic cells occurs predominantly via IgM and is limited to late apoptotic (secondary necrotic) cells.

Apoptotic cells activate complement via various molecular mechanisms. It is not known which of these mechanisms predominate in a physiological environment. Using Jurkat cells as a model, we investigated complement deposition on vital, early and late apoptotic (secondary necrotic) cells in a physiological medium, human plasma, and established the main molecular mechanism involved in this activation. Upon incubation with recalcified plasma, binding of C3 and C4 to early apoptotic cells was similar to background binding on vital cells. In contrast, late apoptotic (secondary necrotic) cells consistently displayed substantial binding of C4 and C3 and low, but detectable, binding of C1q. Binding of C3 and C4 to the apoptotic cells was abolished by EDTA or Mg-EGTA, and also by C1-inhibitor or a monoclonal antibody that inhibits C1q binding, indicating that complement fixation by the apoptotic cells was mainly dependent on the classical pathway. Late apoptotic cells also consistently bound IgM, in which binding significantly correlated with that of C4 and C3. Depletion of plasma for IgM abolished most of the complement fixation by apoptotic cells, which was restored by supplementation with purified IgM. We conclude that complement binding by apoptotic cells in normal human plasma occurs mainly to late apoptotic, secondary necrotic cells, and that the dominant mechanism involves classical pathway activation by IgM.

Animals↗

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↗

PubMedDate not supplied

Mycophenolic acid and methotrexate inhibit lymphocyte cytokine production via different mechanisms.

Mycophenolic acid (MPA) and methotrexate (MTX) are immunosuppressive drugs used for the treatment of various immunological disorders. MPA is an inhibitor of inosine monophosphate dehydrogenase and MTX is a folate antagonist that inhibits tetrahydrofolate reductase. Production of T cell cytokines in whole blood cultures, as well as in PBMC cultures, is inhibited by a low concentration of both drugs. Inhibition of cytokine production after monocyte stimulation was less evident. The mechanism by which inhibition is achieved is different for both drugs. Inhibition of T cell cytokine production by MPA was more profound and started earlier compared to the inhibition by MTX. MTX induced apoptosis in T cells that became activated, whereas MPA prevented activation of T cells by arresting the cell cycle in the G0/G1 phase. Addition of guanosine and adenosine can overcome this cell cycle arrest, even after several days. Furthermore MPA inhibited the expression of activation markers HLA-DR and CD71 on T cells. The observation that MTX cannot prevent T cell activation but induces apoptosis in activated T cells, and that MPA reversibly prevents activation of T cells could explain the immunosuppressive effects of both these drugs.

Apoptosis↗