Wilfried Bautsch

Discrimination of sepsis and systemic inflammatory response syndrome by determination of circulating plasma concentrations of procalcitonin, protein complement 3a, and interleukin-6.

ObjectiveTo evaluate whether plasma concentrations of procalcitonin (PCT), interleukin-6 (IL-6), protein complement 3a (C3a), leukocyte elastase (elastase), and the C-reactive protein (CRP) determined directly after the clinical onset of sepsis or systemic inflammatory response syndrome (SIRS) discriminate between patients suffering from sepsis or SIRS and predict the outcome of these patients. DesignProspective study. SettingMedical intensive care unit at a university hospital. PatientsTwenty-two patients with sepsis and 11 patients with SIRS. Measurements and Main ResultsThe plasma concentrations of PCT, C3a, and IL-6 obtained ≤8 hrs after clinical onset of sepsis or SIRS but not those of elastase or CRP were significantly higher in septic patients ( PCTmedian, 16.8 ng/mL, range, 0.9–351.2 ng/mL, p = .003; C3a: median, 807 ng/mL, range, 422-4788 ng/mL, p < .001; IL-6: median, 382 pg/mL, range, 5–1004 pg/mL, p = .009, all Mann-Whitney rank sum test) compared with patients suffering from SIRS ( PCTmedian, 3.0 ng/mL, range, 0.7–29.5 ng/mL; C3a: median, 409 ng/mL, range, 279–566 ng/mL; IL-6: median, 98 pg/mL, range, 23–586 pg/mL). The power of PCT, C3a, and IL-6 to discriminate between septic and SIRS patients was determined in a receiver operating characteristic analysis. C3a was the best variable to differentiate between both populations with a maximal sensitivity of 86% and a specificity of 80%. An even better discrimination (i.e., a maximal sensitivity of 91% and a specificity of 80%) was achieved when PCT and C3a were combined in a “sepsis score.” C3a concentrations also helped to predict the outcome of patients. Based on the sepsis score, a logistic regression model was developed that allows a convenient and reliable determination of the probability of an individual patient to suffer from sepsis or SIRS. ConclusionsOur data show that the determination of PCT, IL-6, and C3a is more reliable to differentiate between septic and SIRS patients than the variables CRP and elastase, routinely used at the intensive care unit. The determination of PCT and C3a plasma concentrations appears to be helpful for an early assessment of septic and SIRS patients in intensive care.

Identification of a Selective Nonpeptide Antagonist of the Anaphylatoxin C3a Receptor That Demonstrates Antiinflammatory Activity in Animal Models

The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N2-[(2,2-diphenylethoxy)acetyl]-l-arginine (SB 290157), functioned as a competitive antagonist of 125I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC50 of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca2+ mobilization in RBL-C3aR cells and human neutrophils with IC50s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca2+ mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRs. It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.

Cutting Edge: Guinea Pigs with a Natural C3a-Receptor Defect Exhibit Decreased Bronchoconstriction in Allergic Airway Disease: Evidence for an Involvement of the C3a Anaphylatoxin in the Pathogenesis of Asthma

Asthma is a major cause of morbidity worldwide with prevalence and severity still increasing at an alarming pace. Hallmarks of this disease include early-phase bronchoconstriction with subsequent eosinophil infiltration, symptoms that may be mimicked in vivo by the complement-derived C3a anaphylatoxin, following its interaction with the single-copy C3aR. We analyzed the pathophysiological role of the C3a anaphylatoxin in a model of experimental OVA-induced allergic asthma, using an inbred guinea pig strain phenotypically unresponsive to C3a. Molecular analysis of this defect revealed a point mutation within the coding region of the C3aR that creates a stop codon, thereby effectively inactivating gene function. When challenged by OVA inhalation, sensitized animals of this strain exhibited a bronchoconstriction decreased by ∼30% in comparison to the corresponding wild-type strain. These data suggest an important role of C3a in the pathogenesis of asthma and define a novel target for drug intervention strategies.

Pharmacological Targeting of Anaphylatoxin Receptors during the Effector Phase of Allergic Asthma Suppresses Airway Hyperresponsiveness and Airway Inflammation

Airway hyperresponsiveness and airway inflammation are hallmarks of allergic asthma, the etiology of which is crucially linked to the presence of Th2 cytokines. A role for the complement anaphylatoxins C3a and C5a in allergic asthma was suggested, as deficiencies of the C3a receptor (C3aR) and of complement factor C5 modulate airway hyperresponsiveness, airway inflammation, and Th2 cytokine levels. However, such models do not allow differentiation of effects on the sensitization phase and the effector phase of the allergic response, respectively. In this study, we determined the role of the anaphylatoxins on the effector phase of asthma by pharmacological targeting of the anaphylatoxin receptors. C3aR and C5a receptor (C5aR) signaling was blocked using the nonpeptidic C3aR antagonist SB290157 and the neutralizing C5aR mAb 20/70 in a murine model of Aspergillus fumigatus extract induced pulmonary allergy. Airway hyperresponsiveness was substantially improved after C5aR blockade but not after C3aR blockade. Airway inflammation was significantly reduced in mice treated with the C3aR antagonist or the anti-C5aR mAb, as demonstrated by reduced numbers of neutrophils and eosinophils in bronchoalveolar lavage fluid. Of note, C5aR but not C3aR inhibition reduced lymphocyte numbers in bronchoalveolar lavage fluid. Cytokine levels of IL-5 and IL-13 in bronchoalveolar lavage fluid were not altered by C3aR or C5aR blockade. However, blockade of both anaphylatoxin receptors markedly reduced IL-4 levels. These data suggest an important and exclusive role for C5aR signaling on the development of airway hyperresponsiveness during pulmonary allergen challenge, whereas both anaphylatoxins contribute to airway inflammation and IL-4 production.

Rapid quantification of C3a and C5a using a combination of chromatographic and immunoassay procedures

Monoclonal antibodies were isolated which reacted specifically with the complement cleavage products C3a, C3adR, C5a, and C5adR but not with the parent molecules C3 or C5. In both cases the mAbs showed a higher affinity towards the desArg forms. These mAbs were used as capture antibodies in immunoassays for C3a/C3adR and C5a/C5adR. The immunoassays are based on the ABICAP technology which ensures for a rapid measurement. Due to the large binding capacity and the very short diffusion pathways in the gel-matrix the binding equilibrium between capture antibodies and the antigen is reached whilst the sample is flowing through the column. Therefore this test represents an endpoint assay offering the possibility of using a single calibration curve for a large number of measurements. With the C3adR assay concentrations down to 16 ng/ml C3adR can be detected. The lower detection limit of the C5adR assay is 1 ng/ml C5adR. The tests for C3a/C3adR, and C5a/C5adR can be performed in 20 to 25 min and this rapid processing of plasma samples should permit the application of these parameters for diagnostic purposes and patient management.

Epitope mapping of a C5a neutralizing mAb using a combined approach of phage display, synthetic peptides and site-directed mutagenesis.

BACKGROUND The anaphylatoxin C5a is a powerful proinflammatory protein generated on activation of the complement system. Recently, we described an anti-hC5a neoepitope specific mAb, mAb 2925, which was raised against the nonapeptide ISHKDMQLG (C5a-(65-73). This mAb is unique in that it recognizes both hC5a and hC5adesArg, even when it is denatured. It inhibits binding of [125I]C5a to its receptor on Bt2-cAMP differentiated U937 cells. OBJECTIVES To define the epitope of mAb 2925, we used a combined approach of a bacteriophage random octapeptide library, synthetic peptides and site-directed mutagenesis. STUDY DESIGN First a phage peptide library was screened with the anti C5a mAb 2925. Then synthetic peptides were synthesized with respect to the sequence information yielded from the phage approach, and used for binding studies. Site-directed mutagenesis was performed to confirm the results from the mapping experiments. RESULTS AND CONCLUSION Most phages selected by biotinylated Fab 2925 displayed sequences on the minor coat protein which correspond to residues within the C-terminus of human C5a. A first consensus motif comprised amino acids His-Lys or His-Arg, which allowed us to define position 67 and 68 as part of the epitope. A second consensus motif was selected, comprising Arg/Lys-Trp-Trp. This motif did not match any residues within the C5a C-terminus. However, when expressed together with the consensus motif His-Arg, as in HRWWXXXX or in HRXKWWXX, binding of these peptides to Fab 2925 increased as compared to peptides expressing the His-Arg motif only. Thus, the Arg/Lys-Trp-Trp motif serves to stabilize the binding of His-Arg to mAb 2925. Synthetic peptide studies revealed further N-terminal residues Ile65 and Ser66 as part of the epitope. A C5a mutant with an exchange Lys68Glu (C5aGlu68) confirmed the participation of Lys68 as a contact residue within the epitope of mAb 2925. Hence, the epitope recognized by mAb 2925 is linear and comprises residues Ile65, Ser66, His67, and Lys68. Thus, we could demonstrate for the first time that a mAb inhibits C5a receptor binding through specific interaction with receptor binding residues of the ligand.

Molecular and phenotypic identification of the yeast pathogen Candida dubliniensis

Abstract Candida dubliniensis is an emerging yeast pathogen generally misclassified as Candida albicans by standard diagnostic procedures. This study examined the efficiency of molecular identification, based on a discriminative PCR test, in a prospective study on the prevalence of C. dubliniensis among 103 oropharyngeal isolates from HIV-infected individuals or transplant recipients, and 30 vaginal isolates. All of the isolates had been classified as C. albicans by standard laboratory procedures. The PCR was evaluated in a blinded fashion against classification achieved by sequencing rDNA. Sequencing results corresponded 100% to the results of the discriminative PCR, indicating the validity of this rapid test. Twenty-one C. dubliniensis isolates were identified, all of them from HIV-infected individuals (prevalence 30%). The internal transcribed spacer regions of the C. dubliniensis isolates were sequenced. Phenotypic features of C. dubliniensis, namely abundant chlamydospore formation, atypical color on CHROMagar, growth defect at 45°C, and colony morphology on Staib agar, were evaluated in a blinded fashion with respect to their discriminative potential, facilitating the design of further epidemiological studies. Carbohydrate assimilation patterns were determined for C. dubliniensis with a novel automated system showing that, in contrast to previous reports, C. dubliniensis is able to utilize D-xylose and trehalose. In evaluating these tests we present a rational approach to identification of the new species and characterization of C. dubliniensis isolates.

Chimeric Receptors of the Human C3a Receptor and C5a Receptor (CD88)

Chimeras were generated between the human anaphylatoxin C3a and C5a receptors (C3aR and C5aR, respectively) to define the structural requirements for ligand binding and discrimination. Chimeric receptors were generated by systematically exchanging between the two receptors four receptor modules (the N terminus, transmembrane regions 1 to 4, the second extracellular loop, and transmembrane region 5 to the C terminus). The mutants were transiently expressed in HEK-293 cells (with or without Gα-16) and analyzed for cell surface expression, binding of C3a and C5a, and functional responsiveness (calcium mobilization) toward C3a, C5a, and a C3a as well as a C5a analogue peptide. The data indicate that in both anaphylatoxin receptors the transmembrane regions and the second extracellular loop act as a functional unit that is disrupted by any reciprocal exchange. N-terminal substitution confirmed the two-binding site model for the human C5aR, in which the receptor N terminus is required for high affinity binding of the native ligand but not a C5a analogue peptide. In contrast, the human C3a receptor did not require the original N terminus for high affinity binding of and activation by C3a, a result that was confirmed by N-terminal deletion mutants. This indicates a completely different binding mode of the anaphylatoxins to their corresponding receptors. The C5a analogue peptide, but not C5a, was an agonist of the C3aR. Replacement of the C3aR N terminus by the C5aR sequence, however, lead to the generation of a true hybrid C3a/C5a receptor, which bound and functionally responded to both ligands, C3a and C5a.

Human C5a anaphylatoxin : gene cloning and expression in Escherichia coli

A gene coding for the human anaphylatoxin C5a was cloned and expressed in Escherichia coli. A combination of reverse transcription of mRNA of the U937 cell line with subsequent preparative polymerase chain reaction was employed to obtain the gene. The sequence was cloned into the plasmid vector pKK 233-2 behind an ATG initiation codon under the control of a trc promotor. After purification by ion exchange chromatography and reversed phase FPLC a mixture of predominantly non-glycosylated recombinant human C5a with a beta-mercaptoethanol adduct at cysteine 27 and the N-methionyl derivative was obtained which was homogeneous on silver-stained gels, immunoreactive with C5a-specific monoclonal antibodies and functionally active in releasing myeloperoxidase from human granulocytes and ATP from guinea pig platelets. The final yield was about 0.4-0.8 mg purified recombinant C5a per liter bacterial culture.

Guinea pig C3 specific rabbit single chain Fv antibodies from bone marrow, spleen and blood derived phage libraries.

We constructed combinatorial immunoglobulin libraries from the whole rabbit antibody repertoire of bone marrow, spleen and peripheral blood of a rabbit immunized with guinea pig complement protein C3. By means of the phage display technology we selected guinea pig C3 specific single chain Fv (scFv) antibodies from each of the libraries. None of the scFv antibodies cross reacted with guinea pig C3a, human C3 or rat C3. The frequency of bone marrow derived C3 positive clones was much higher as compared to blood or spleen derived clones. Additionally bone marrow and spleen derived clones show higher diversity than clones, obtained from blood, as determined by fingerprint analysis with the restriction enzyme AluI. Dissociation rate constants for all scFvs were similar, indicating that the source of the scFvs had no influence on affinities. The antibody fragments were used to analyze complement activation during xenotransplantation. Several blood or bone marrow derived scFvs bound to C3 located on rat liver endothelium after hyperacute rejection of a heterotopically transplanted rat liver into guinea pig. These data demonstrate that monoclonal rabbit scFvs can be easily generated from recombinant phage display libraries, constructed from spleen, blood or bone marrow. The selected guinea pig C3 specific scFvs appear to be useful to detect complement activation during xenotransplantation in guinea pigs.