A Regional health care network: eHealth.Braunschweig. Domain fields and architectural challenges.

BACKGROUND: Health care network eHealth.Braunschweig has been started in the South-East region of Lower Saxony in Germany in 2009. It composes major health care players, participants from research institutions and important local industry partners. OBJECTIVES: The objective of this paper is firstly to describe the relevant regional characteristics and distinctions of the eHealth.Braunschweig health care network and to inform about the goals and structure of eHealth.Braunschweig; secondly to picture and discuss the main concepts and domain fields which are addressed in the health care network; and finally to discuss the architectural challenges of eHealth.Braunschweig regarding the addressed domain fields and defined requirements. METHODS: Based on respective literature and former conducted projects we discuss the project structure and goals of eHealth.Braunschweig, depict major domain fields and requirements gained in workshops with participants and discuss the architectural challenges as well as the architectural approach of eHealth.Braunschweig network. RESULTS: The regional healthcare network eHealth.Braunschweig has been established in April 2009. Since then the network has grown constantly and a sufficient progress in network activities has been achieved. The main domain fields have been specified in different workshops with network participants and an architectural realization approach for the transinstitutional information system architecture in the healthcare network has been developed. However, the effects on quality of information processing and quality of patient care have not been proved yet. Systematic evaluation studies have to be done in future in order to investigate the impact of information and communication technology on the quality of information processing and the quality of patient care. CONCLUSIONS: In general, the aspects described in this paper are expected to contribute to a systematic approach for the establishment of regional health care networks with lasting and sustainable effects on patient-centered health care in a regional context.

Prevalence and molecular epidemiology of meticillin-resistant Staphylococcus aureus in nursing home residents in northern Germany.

Nursing home residents are a population at risk for carrying meticillin-resistant Staphylococcus aureus (MRSA). To better guide infection control and healthcare network initiatives, we investigated the point prevalence and molecular epidemiology of MRSA colonisation among nursing home residents in Brunswick, northern Germany. Among the 32 participating nursing homes of the available 34 in the region, 68% of residents (1827 of 2688) were screened for nasal and/or wound colonisation. A total of 139 residents (7.6%; 95% confidence interval: 6.4-8.8%) were identified as MRSA positive, almost six-fold more than the 24 MRSA carriers (0.9%) expected according to the nursing homes' pre-test information. Although known risk factors including urinary tract catheters, wounds, preceding hospital admission, and high grade resident care were confirmed, none was sensitive enough to be considered as the sole determinant of MRSA carriage. spa typing revealed that more than 70% of isolates belonged to the Barnim strain (ST-22, EMRSA-15, CC22) typical for hospital-acquired MRSA in northern Germany. There was no evidence for the presence of community-acquired or livestock-associated S. aureus strains. These data show that in northern Germany MRSA has spread from the hospital environment to other healthcare institutions, which must now be regarded as important reservoirs for MRSA transmission.

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, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB 290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s 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 Ca(2+) 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 approximately 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.

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

OBJECTIVE: To 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. DESIGN: Prospective study. SETTING: Medical intensive care unit at a university hospital. PATIENTS: Twenty-two patients with sepsis and 11 patients with SIRS. MEASUREMENTS AND MAIN RESULTS: The plasma concentrations of PCT, C3a, and IL-6 obtained < or =8 hrs after clinical onset of sepsis or SIRS but not those of elastase or CRP were significantly higher in septic patients (PCT: median, 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 (PCT: median, 3.0 ng/mL, range, 0.7-29.5 ng/mL; C3a: median, 409 ng/mL, range, 279566 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. CONCLUSIONS: Our 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.

A novel computational method for predicting the transmembrane structure of G-protein coupled receptors: application to human C5aR and C3aR.

A novel algorithm was applied to the sequences of bacteriorhodopsin (BRh), of rhodopsin (Rh), and of the two human anaphylatoxin receptors, C5a-receptor (hC5aR) and C3a-receptor (hC3aR), that predicts their transmembrane domains (TMD) according to energy criteria alone, on the basis of their sequences and a template structure for each. Two consecutive criteria were applied for the predictions: the first is hydrophobicity of a sequence of residues, which determines the candidate stretches of residues that form one of the transmembrane helices. The second criterion is an energy function composed of inter residue contact energies, of hydrophobic contributions due to membrane exposure and of the interactions of a few residues with the phospholipid head groups. The sequence of candidate residues for each helix is longer than that of the template, and is finally determined by threading each of the candidate stretches on each of the template helices and evaluating the energy for all possible configurations. Contact energies between residues were taken from a database (Miyazawa S and Jernigan RL (1996) J Mol Biol 256 623-44). The algorithm predicts well the TMD structure of BRh based on its own template, and the TMD structure of Rh conforms well with the model of Baldwin et al (Baldwin JM Schertler GFX and Unger VM (1997) J Biol Chem 272 144-64). Results for the construction of the TMD of hC5aR and hC3aR were compared, employing the template structure of Rh. Most of the results for these receptors are in accord with alignments and with mutation experiments on hC5aR and hC3aR. The predictions may serve as a basis for future mutagenesis experiments of these receptors.

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.

Molecular and phenotypic identification of the yeast pathogen Candida dubliniensis.

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 degrees 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.

Receptor activation by human C5a des Arg74 but not intact C5a is dependent on an interaction between Glu199 of the receptor and Lys68 of the ligand.

Despite the expression of only one type of receptor, there is great variation in the ability of different cell types to discriminate between C5a and its more stable metabolite, C5a des Arg74. The mechanism that underlies this phenomenon is not understood but presumably involves differences in the interaction with the C5a receptor. In this paper, we have analyzed the effects of a substitution mutation of the receptor (Glu199 --> Lys199) and the corresponding reciprocal mutants (Lys68 --> Glu68) of C5a, C5a des Arg74 and peptide analogues of the C-terminus of C5a on the ability of the C5a receptor to discriminate between ligands with and without Arg74. The use of these mutants indicates that the Lys68/Glu199 interaction is essential for activation of receptor by C5a des Arg74 but not for activation by intact C5a. The substitution of Asp for Arg74 of C5a [Lys68] produces a ligand with equal potency on both the wild-type and mutant receptors, suggesting that it is the C-terminal carboxyl group rather than the side chain of Arg74 that controls the responsiveness of the receptor to Lys68. In contrast, the mutation of Lys68 to Glu(68) has little effect on the ability of either C5a or C5a des Arg(74) to displace [(125)I]C5a from the receptors, indicating that binding of ligand and receptor activation are distinct but interdependent events. C5a and the truncated ligand, C5a des Arg74, appear to have different modes of interaction with the receptor and the ability of the human C5a receptor to discriminate between these ligands is at least partly dependent on an interaction with the receptor residue, Glu199.

Selection of a C5a receptor antagonist from phage libraries attenuating the inflammatory response in immune complex disease and ischemia/reperfusion injury.

A C5a-receptor antagonist was selected from human C5a phage display libraries in which the C terminus of des-Arg74-hC5a was mutated. The selected molecule is a competitive C5a receptor antagonist in vitro and in vivo. Signal transduction is interrupted at the level of G-protein activation. In addition, the antagonist does not cause any C5a receptor phosphorylation. Proinflammatory properties such as chemotaxis or lysosomal enzyme release of differentiated U937 cells, as well as C5a-induced changes in intracellular Ca2+ concentration of murine peritoneal macrophages, are inhibited. The in vivo efficacy was evaluated in three different animal models of immune complex diseases in mice, i.e., the reverse passive Arthus reaction in the peritoneum, skin, and lung. The i.v. application of the C5a receptor antagonist abrogated polymorphonuclear neutrophil accumulation in peritoneum and markedly attenuated polymorphonuclear neutrophil migration into the skin and the lung. In a model of intestinal ischemia/reperfusion injury, i.v. administration of the C5a receptor antagonist decreased local and remote tissue injury: bowel wall edema and hemorrhage as well as pulmonary microvascular dysfunction. These data give evidence that C5a is an important mediator triggering the inflammatory sequelae seen in immune complex diseases and ischemia/reperfusion injury. The selected C5a receptor antagonist may prove useful to attenuate the inflammatory response in these disorders.

Analysis of the C5a anaphylatoxin core domain using a C5a phage library selected on differentiated U937 cells.

The human anaphylatoxin C5a is a 74-amino acid comprising polypeptide with a plethora of biological functions. Site directed mutagenesis studies suggest that several residues within the core and the C-terminus mediate the interaction with the C5a receptor. However, the contribution of particular core residues to receptor binding remained to be clarified. By means of the phage display technique, the loop between positions 35-40 was randomly mutated and the resulting C5a[35-40] fusion phage library affinity selected on C5a receptor expressing U937 cells. After five rounds of affinity enrichment, residues Arg37 and Arg40 were preferably selected. Enrichment was as high as 100% for Arg37 and 79% for Arg40. No significant enrichment of consensus residues could be obtained for positions 35, 36, 38 and 39. The core mutant C5a[A35E36R37A38S39R40], in which only Arg37/40 and Ala38 are of the native C5a sequence, was as potent as native C5a in both receptor binding and enzyme release examined on U937 cells. In contrast, replacement of Arg40 as in the mutant C5a[Q35E36R37I38L39N40] resulted in a 10-fold decrease in both binding and functional activities. Thus, selected out of a multiplicity of possibilities by the natural binding partner, Arg37 as well as Arg40 appear to be anchor residues in binding to the C5a receptor.

Modulation of C3a activity: internalization of the human C3a receptor and its inhibition by C5a.

The C3a receptor (C3aR) is expressed on most human peripheral blood leukocytes with the exception of resting lymphocytes, implying a much higher pathophysiological relevance of the anaphylatoxin C3a as a proinflammatory mediator than previously thought. The response to this complement split product must be tightly regulated in situations with sustained complement activation to avoid deleterious effects caused by overactivated inflammatory cells. Receptor internalization, an important control mechanism described for G protein-coupled receptors, was investigated. Using rabbit polyclonal anti-serum directed against the C3aR second extracellular loop, a flow cytometry-based receptor internalization assay was developed. Within minutes of C3a addition to human granulocytes, C3aR almost completely disappeared from the cell surface. C3aR internalization could also be induced by PMA, an activator of protein kinase C. Similarly, monocytes, the human mast cell line HMC-1, and differentiated monocyte/macrophage-like U937-cells exhibited rapid agonist-dependent receptor internalization. Neither C5a nor FMLP stimulated any cross-internalization of the C3aR. On the contrary, costimulation of granulocytes with C5a, but not FMLP, drastically decreased C3aR internalization. This effect could be blocked by a C5aR-neutralizing mAb. HEK293-cells transfected with the C3aR, with or without Galpha16, a pertussis toxin-resistant G protein alpha subunit required for C3aR signal transduction in these cells, did not exhibit agonist-dependent C3aR internalization. Additionally, preincubation with pertussis toxin had no effect on C3a-induced internalization on PMNs. C3aR internalization is a rapid negative control mechanism and is influenced by the C5aR pathway.

Cutting edge: Fc receptor type I for IgG on macrophages and complement mediate the inflammatory response in immune complex peritonitis.

The contributions of Fc receptors (FcRs) for IgG (FcgammaRs) and complement to immune complex (IC)-mediated peritonitis were evaluated in BALB/c-, C57BL/6-, FcRgamma chain-, and FcR type III for IgG (FcgammaRIII)-deficient mice, backcrossed to the C57BL/6 background. In BALB/c mice, but not in C57BL/6 mice, neutrophil migration was markedly attenuated after complement depletion. In mice lacking FcRgamma chain, neutrophil migration was abolished, whereas it was unaffected in FcgammaRIII-deficient mice. Huge amounts of TNF-alpha (TNF) were found in the peritoneal exudate of BALB/c and C57BL/6 mice but were absent in mice lacking FcRgamma chain or FcgammaRIII. Surprisingly, a functional inhibition of TNF in BALB/c and C57BL/6 mice had no effect on neutrophil infiltration. These data provide evidence that in IC peritonitis, the activation of FcR type I for IgG on peritoneal macrophages and the activation of the complement cascade, but not the interaction of ICs with FcgammaRIII and the subsequent release of TNF, initiate the inflammatory response in BALB/c and C57BL/6 mice.

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 Galpha-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.

Detection and typing of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks attached to human skin by PCR.

Live Ixodes ricinus ticks attached to humans residing in Germany were examined for borreliae by dark-field microscopy and PCR. Borrelia species were identified by 16S rRNA sequence analysis, which showed the presence of several species, some not yet defined, and a high prevalence of multiply infected ticks.

Human anaphylatoxin C4a is a potent agonist of the guinea pig but not the human C3a receptor.

The interaction of human anaphylatoxin C4a with the guinea pig (gp) and human (hu) C3a receptors (C3aR) was analyzed using human rC4a, which exhibited C4a-specific activity on guinea pig platelets. A gpC3aR of 475 residues with a large second extracellular loop and a peptide sequence approximately 60% identical to the huC3aR was isolated from a genomic DNA library and found to be expressed in guinea pig heart, lung, and spleen. HEK-293 cells cotransfected with this clone, and a cDNA encoding G alpha-16 specifically bound (Kd = 1.6+/-0.7 nM) and responded functionally to C3a with an intracellular calcium mobilization (ED50 = 0.18+/-0.02 nM). Human rC4a weakly bound to both the hu- and gpC3aR (IC50 > 1 microM). However, only HEK-293 cells expressing the gpC3aR responded functionally to rC4a (ED50 = 8.7+/-0.52 nM), while cells expressing the huC3aR did not (c < or = 1 microM). Thus, through an interaction with the C3aR, huC4a may elicit anaphylatoxic effects in guinea pigs but not in man.

Genomic organization of the human C3a receptor.

The human C3a receptor (C3aR) mediates the activation of cells by the potent proinflammatory chemoattractant C3a, an anaphylatoxin, generated in the early phase of an inflammatory reaction by proteolytic cleavage of the complement component C3. To understand the molecular mechanisms that regulate C3aR gene expression, we initiated studies to determine its genomic and mRNA organization. We now report the following novel findings: (1) The C3aR is a single-copy gene as shown by Southern hybridization of human genomic DNA. (2) Using PCR amplification of DNA from monochromosomal somatic cell hybrid and radiation hybrid panels, the C3aR locus was mapped to chromosome 12p13. (3) Genomic DNA clones encompassing the C3aR locus were isolated from a human genomic DNA library and characterized by restriction mapping, Southern blotting, PCR analysis and DNA sequencing. Comparison of the genomic with the known cDNA sequences revealed a single 6-kb intron sequence located 1 1 bp upstream of the ATG initiation codon. The open reading frame and the complete 3' untranslated region are encoded on a single exon.

Comparison of the genome organization of pathogenic neisseriae.

Current efforts to completely sequence the meningococcal and gonocococcal genomes raise the question whether the lessons learned from the sequenced strains may be safely extrapolated to other members of these species, or whether, in view of the fact that Neisseriae are highly recombinogenic and exhibit a high degree of horizontal intra- and interspecies genetic transfer, only clone-specific conclusions are valid. From the known physical and genetic maps of each of two gonococcal and meningococcal strains, it would appear that both species exhibit a species-specific conservation in their genetic organization while the interspecies comparison revealed several rearrangements, although still with a high overall similarity. However, these data contrast with other evidence suggesting intra-species rearrangements, such as the nonconserved I-CeuI macrorestriction patterns of different meningococcal and other neisserial strains. Since I-CeuI cuts within the 23S-rRNA sequence, the restriction pattern should give reliable information on the distribution of rrn loci in the neisserial genomes. Further studies are warranted to answer these questions.

A detailed analysis of the C5a anaphylatoxin effector domain: selection of C5a phage libraries on differentiated U937 cells.

We have used a phage-display-based system to investigate the effect of simultaneous substitutions within the C5a effector domain. Two different libraries were constructed. In library I, known binding positions 67, 68, 72 and 74 of human complement C5a (hC5a) and in library II, positions 69-73 of hC5a without C-terminal Arg74 (des-Arg74-C5a) were randomly mutated. In more than 80% (position 72) or 90% (positions 68 and 74) of all cases, the original residues of hC5a were selected from library I, demonstrating that the phage system can be used to define binding points within the C5a molecule. Surprisingly, in more than 90% of all clones, a Phe residue was enriched at position 67 instead of the original His residue which, however, did not affect the binding affinity or the signalling activity. In library II, Leu was preferentially selected at positions 70-72 and Tyr at position 73, while no enrichment of an individual amino acid was observed at position 69. Mutants with (a) Leu in positions 71 and 72 (b) Ser or Leu in position 70 and (c) Arg or Tyr in position 73, showed a 4-10-fold higher binding affinity as compared to des-Arg74-[Ala27, Phe67]C5a. The binding affinity was indistinguishable from that of hC5a. In consequence, not only position 72 but also positions 70, 71 and 73 are able to interact with the C5a receptor, whereas position 69 is not. Intriguingly, one mutant with a high binding affinity but without signalling activity was selected. Thus, random mutagenesis of phage-displayed C5a was proven to be a powerful strategy to define receptor-binding points and to select C5aR antagonists based on the structure of the natural ligand.