Biomedical subjects
Publications and source records attributed to W Bautsch.
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.
The pathophysiological relevance of the complement split product C3a as a proinflammatory mediator is still ill defined. The expression pattern of the human C3a receptor (C3aR) can provide important clues for the role of this anaphylatoxin in inflammation. There is strong evidence for C3aR expression on basophils, and eosinophils, but additionally, only on tumor cell lines of leukemic or hepatic origin. It is unclear whether neutrophils also express the C3aR, but need a costimulus provided by eosinophils for certain biological responses, or whether neutrophils lack the C3aR and respond to C3a via a secondary stimulus generated by eosinophils, i.e., by an indirect mode. In the present study, polyclonal antiserum raised against the second extracellular loop of the C3aR was used to characterize C3aR expression on peripheral blood leukocytes. For high degree purification of neutrophils, a negative selection method was established that decreased the contamination with CD9(bright+) eosinophils down to <0.2%. Flow cytometric analyses, functional assays, and binding assays on highly purified neutrophils confirmed C3aR expression and coupling. Monocytes were identified as an additional C3aR-positive cell population of the peripheral blood. The expression of the C3aR on eosinophils could be confirmed. In contrast, the receptor could not be detected on unchallenged B or T lymphocytes (or lymphocyte-derived Raji cells).
Binding and effector domains of the human anaphylatoxin C5a have been determined by either site directed mutagenesis or synthetic peptide studies. However, the lack of specific selection methods, which allow direct investigation of C5a-C5a-receptor interaction made these studies laborious. To overcome these limitations we have constructed a novel Fos-C5a expressed on the tip of a filamentous phage. To guarantee for a free C-terminus which is required for C5a activity C5a cDNA was cloned into the phagemid vector pJuFo. Helper phage infection of pJuFc-C5a transformed cells resulted in a mutant phage displaying Fos-C5a on its surface. However studies with Bt2cAMP differentiated U937 cells revealed that phage displayed Fos-C5a is functional inactive. Subsequently we replaced a nonconserved cysteine residue at position 27 by alanine and obtained Fos-C5aAla27. Both the purified and the phage displayed Fos-C5aAla27 proteins were functional active and induced enzyme release from differentiated U937 cells. In addition, purified Fos-C5aAla27 exhibited the same binding profile as compared to rhC5a. Fos-C5aAla27 displaying phages were mixed with phage harboring only the pJuFo plasmid at a ratio of 10(6). After four successive rounds of panning on differentiated U937 cells Fos-C5aAla27 phages were enriched to 100% as shown by C5a-specific ELISA. We expect this approach to prove helpful for studying C5a-C5a-receptor interactions. i.e. to screen C5a libraries for high affinity binders with agonistic or antagonistic properties directly on cells.
The biological effects of the potent inflammatory mediator C5a, a complement split product, on human neutrophils and monocytes are limited by the rapid internalization of its specific receptor (C5aR, CD88). The C terminus of the C5aR is phosphorylated after stimulation with C5a of phorbol ester, and this phosphorylation might lead to receptor internalization. In this context, we have studied the effects on C5aR internalization of C5a, phorbol 12-myristate 13-acetate (PMA), the protein kinase inhibitor staurosporine, and pertussis toxin on rat basophilic RBL.2H3 cells stably transfected with the human wild-type or mutant C5aR. C5aR mutants lacked either part of the cytosolic C terminus, including suggested major phosphorylation sites, or a putative phosphorylation motif for protein kinase C in the third cytosolic loop. Additionally, agonist-induced internalization was analyzed on HEK293 cells co-transfected with C5aR and the pertussis toxin-resistant G protein alpha subunit, G alpha 16. Staurosporine-sensitive agonist-dependent C5aR internalization could be detected, suggesting that C5aR phosphorylation, most likely of the C terminus, participates in this type of internalization. In contrast, PMA-induced C5aR internalization seems to be independent of putative phosphorylation sites in either the truncated section of the C terminus or the third cytosolic loop. The phorbol ester-induced C5aR internalization may, therefore, be caused by an indirect and less specific effect of protein kinase C on the internalization machinery. Manipulation of the pertussis toxin-sensitive or -resistant G protein-dependent signal transduction had no effect on ligand-induced internalization.
OBJECTIVE: To investigate protein complement 3a (C3a) and protein complement 3 (C3) plasma levels in trauma patients directly after the injury, in relation to the patients' outcome, the development of sepsis, or the injury severity, as determined by either the Polytrauma Score (PTS), the Injury Severity Score (ISS), or the Trauma and Injury Severity Score (TRISS). DESIGN: Prospective study. SETTING: Surgical intensive care unit in a university hospital. PATIENTS: Thirty-four patients with multiple trauma. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: C3a and C3 concentrations, as well as the C3a/C3 ratio, were determined at the time of the accident (T0), at the emergency admission (T1), 8 hrs after the accident (T2), and every 8 hrs until day 3, every 12 hrs until day 6, and once daily on days 7 and 8. The C3a plasma concentrations and the C3a/C3 ratios of nonsurvivors were significantly greater at T0 or T1 as compared with those survivors (p = .008 or .033). Patients who developed sepsis had higher C3a plasma levels at the scene of accident than patients without complications. However, these differences did not reach statistical significance (p = .051), although a clear trend was apparent. Patients were grouped according to the severity of injury, as determined by either the PTS, ISS, or TRISS. We found significant differences in the both the mean C3a values and the C3a/C3 ratio among the different groups, during the first 8 hrs after the injury. In addition, a significant correlation was observed between the C3a concentration or the C3a/C3 ratio at T0 to T2 and either the ISS (r2 = .49), PTS (r2 = .22) or the TRISS (r2 = .45), which was similar to correlations between injury severity scores themselves (r2 = .36 to .58). CONCLUSIONS: Complement activation occurs immediately after the injury. The degree of activation is a hallmark for the outcome of a patient. Determination of C3a concentrations, at the scene of the accident, may prove helpful to assess the severity of the injury and to determine the prognosis. The amount of C3a and the C3a/C3 ratio may be useful as additional parameters to the existing trauma scoring systems, such as, PTS, ISS, and TRISS.
We have analyzed the induction of the receptor for the anaphylatoxic peptide C3a (C3aR) by the immunomodulator IFN-gamma, the phorbol ester PMA, and dibutyryl cAMP (Bt2cAMP) in comparison with the C5a receptor (C5aR, CD88). For U937 cells, IFN-gamma and Bt2cAMP up-regulated the C3aR to the same extent, whereas Bt2cAMP was 20-fold more effective in C5aR induction. PMA increased the expression of the C5aR, and acted synergistically with IFN-gamma. In contrast, PMA did not increase specific 125I-hC3a binding, and actually antagonized C3aR induction by IFN-gamma. Two related human cell lines of the myeloblastic/monocytic lineage, HL-60 and Mono Mac 6, showed inducibility of the C3aR similar to U937 cells. The two receptors showed subtle differences in signal transduction. Despite comparable numbers of both receptors, IFN-gamma potentiated activation of the C5aR but not the C3aR, as measured by an increase in free cytosolic Ca2+ upon ligand activation. Interestingly, Bt2cAMP-treatment led to a functional response to C3a in U937 cells. Such differences in receptor regulation and signaling might underlie the partly differing physiologic effects of C3a and C5a on, for example, chemotaxis, induction of oxidative burst, or immunoregulatory functions.
The human C5a receptor (C5aR) belongs to the family of G-protein-coupled receptors with seven transmembrane helices. This part of the molecule is thought to contain part of the ligand-binding pocket, specifically to bind the C-terminal Arg of human C5a. Guided by sequence similarity and molecular modelling studies, several residues including polar (Asn119, Thr168, Gln259) as well as all conserved charged amino acids in the upper transmembrane region of the C5aR (Asp37, Asp82, Arg175, Arg2O6, Asp282) were exchanged by site-directed mutagenesis. Receptor mutants were transiently expressed in COS cells and analyzed for altered binding behaviour and/or localization at the cell surface by immunofluorescence. For all residues, suitable mutants could be found that exhibited wild-type affinity towards the ligand, providing evidence against a major contribution of these residues to high-affinity ligand binding. Some mutants, however, exhibited a complete (Asp282-->Ala) or partial loss of ligand-binding capacity (Arg175-->Ala, Arg2O6-->Gln) despite adequate expression levels on the cell surface. This phenotype was further analyzed in the [Gln2O6]C5aR mutant: quantitative flow cytometric analysis of epitope-tagged receptor derivatives in 293 cells confirmed an equal level of wild-type and mutant C5aR on the cell surface. Competitive binding curves revealed the presence of only a small population (<10%) of high-affinity sites (Kd approximately 2 nM), which was functionally active at 20 nM in the heterologous Xenopus oocyte expression system after coexpression of G alpha-16. The number of high-affinity sites of wild-type and [Gln2O6]C5aR in 293 cells could be up-regulated by coexpression of Gi alpha-2 and down-regulated by GTP[gamma S]-mediated uncoupling of the G-protein receptor interaction in membrane preparations. These findings are compatible with a model in which the Arg2O6 residue located in the upper third of transmembrane helix V determines high-affinity binding in the human C5aR by affecting the intracellular G-protein coupling.
A cDNA clone encoding the human C3a anaphylatoxin receptor (C3aR) was isolated from a pcDNAI/Amp expression library prepared from U-937 cells which had been differentiated with dibutyryl cAMP to a macrophage-like phenotype. The cDNA clone contained an insert of 4.3 kbp and was able to confer to transfected human HEK-293 cells the capacity to bind specifically iodinated human C3a. Chinese hamster ovary cells co-transfected with this cDNA clone and a G-protein alpha subunit (G alpha-16) became functionally responsive to C3a and a C3a analog synthetic peptide, as measured by increased phosphoinositide hydrolysis. As inferred from the cDNA sequence, the clone encodes a 482-residue polypeptide with seven hydrophobic membrane-spanning helices and a high homology to the human C5a and formyl-Met-Leu-Phe receptors. Uniquely among the family of G-protein coupled receptors, the C3aR contains an exceptionally large second extracellular loop of approximately 175 residues. Northern hybridizations revealed an approximately 2.3-kb transcript as the major and an additional approximately 3.9 kb-transcript as a minor transcription product of the C3aR. The C3aR appears to be widely expressed in different lymphoid tissues, as shown by Northern hybridizations, providing evidence for a central role of the C3a anaphylatoxin in inflammatory processes.
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.
A physical map of the chromosome of Neisseria meningitidis B1940 has been constructed by one- and two-dimensional pulsed-field gel electrophoresis techniques. Complete macrorestriction maps for the enzymes Nhel (16 sites), Sgfl (13 sites), Sfil (11 sites) and l-Ceul (4 sites), as well as a partial restriction map for the restriction enzyme Spel (15 of c. 28 sites) could be established. Altogether, 59 restriction sites were mapped on a single circular chromosome of 2.3 Mbp. By restriction endonuclease digestion and Southern hybridization of cloned genetic markers, 39 genetic loci were assigned to this map. Comparison with the metabolic maps of Neisseria gonorrhoeae MS11-N198 and FA1090 revealed a high degree of conservation in the arrangement of gene loci among these two species, although four out of 24 genetic loci are located at different chromosomal positions, indicating several genomic rearrangements.
The systemic inflammatory response of the body to invading microorganisms, termed sepsis, leads to profound activation of the complement system. Pathophysiological concepts suggest that complement activation occurs very early in this syndrome. Thus, we discuss whether the determination of concentrations of the complement components C3a, C5a, and C3 in plasma as well as of the C3a/C3 ratio might be helpful to diagnose sepsis early. For this purpose, 33 patients from an intensive care unit were monitored for 10 days. In comparison with healthy donors, C3a levels and the C3a/C3 ratio of intensive-care-unit patients were significantly elevated (P < 0.0001) on admission. In contrast, C3 levels were significantly reduced (P < 0.0001) but increased during the study. C5a levels in the plasma of healthy donors and patients were identical. Twenty-two of 33 patients fulfilled microbiological and clinical criteria of sepsis. Eleven patients had signs of systemic inflammatory response syndrome but no microbiological evidence of sepsis. The groups could be differentiated from each other by their C3a levels or their C3a/C3 ratios during the first 24 h after the clinical onset of sepsis (P < 0.05). Septic patients in shock had higher C3a levels than normotensive septic patients, although the differences were not significant. Nonsurvivors had significantly higher C3a levels on admission than survivors (P = 0.0185). No differences were found between septic patients who developed adult respiratory distress syndrome and those who did not. Thus, determination of C3a concentrations in plasma may prove useful (i) to diagnose sepsis early, (ii) to differentiate between patients with sepsis and those with systemic inflammatory response syndrome, and (iii) to assess prognosis.
The insertion element IS1301 has been shown to mediate capsule phase variation in Neisseria meningitidis found in N. serogroup B by reversible insertional inactivation of the siaA gene. We have determined the target site specificity of this element by cloning and sequencing the insertion sites of 12 identical IS1301 copies found in N. meningitidis B1940. A target consensus core of 5'-AYTAG-3' was identified, with the central TA being duplicated following insertion. Additional features around the target sites, including extended palindromic symmetry, stem-loop formation, and the high incidence of AT tracts, indicate that other factors, such as DNA secondary structure, are involved in target recognition. The left inverted repeat of an IS1016-like element acts as a hot spot for insertion, with one insertion element combination located upstream of their gene. According to further sequence analysis, we were able to place IS1301 in the IS5 subgroup within the IS4 family of elements. A survey of 135 Neisseria strains indicated the presence of IS1301 in 27.9 to 33.3% of N. meningitides serogroup B, C, and W135 strains and in 86.7% of serogroup Y strains. IS1301 did not occur in serogroup A strains, in Neisseria gonorrhoeae, or in apathogenic Neisseria spp.
The human leukocyte chemoattractant receptors for complement factor C5a (C5a-R) and N-formylated peptides (fMLF-R) are important members of the superfamily of G-protein coupled receptors (GPCR). Uniquely among the GPCR, these two receptors cannot be expressed in a functionally active form in the oocytes of the frog Xenopus laevis, but require substitution of total RNA of the myelomonocytic U-937 or HL-60 cell lines, respectively. Recently, it was reported that the C5a-R may couple to the alpha subunit of G-16. We have tested this G-protein for its ability to complement the signal transduction cascade of the C5a-R and fMLF-R in Xenopus oocytes. Injection of cRNA for the C5a-R in combination with G alpha-16 led to expression of a functional C5a-R as measured by ligand-induced whole cell current. In contrast to a previous report, the fMLF-R exhibited some residual functional activity when transiently expressed in Xenopus oocytes the extent of which could, however, substantially be increased by coexpression of G alpha-16. Thus, G alpha-16 complements the signal transduction cascade of both receptors in Xenopus laevis oocytes and is most likely the complementing factor present in the U-937 and HL-60 cell lines.
On human mature monocytes the immunomodulator IFN-gamma has been shown to down-regulate the receptor for the anaphylatoxic peptide C5a (CD88, C5aR). In this study, we show that in immature myelo-/monoblastic U937, HL60, and MonoMac6 cells, IFN-gamma induces C5aR-ligand binding activity. In U937 cells, this induction cannot be blocked by the protein kinase C inhibitor staurosporine. An increase in free cytosolic Ca2+ upon ligand binding indicates functional coupling of this receptor in U937 and HL-60 cells. G-Proteins involved in this C5a responsiveness after IFN-gamma induction are completely pertussis toxin sensitive. Our data suggest that an additional pertussis toxin-resistant pathway exists in U937 cells after induction by dibutyryl cAMP. However, this is not due to changes in the mRNA level of the pertussis toxin-insensitive G-protein subunit G alpha 16. Induction by dibutyryl cAMP, but not that by IFN-gamma, resulted in C5a-dependent release of N-acetyl-beta-D-glucosaminidase, further highlighting functional differences in the effects of the inducers. Our data show an IFN-gamma-dependent increase in C5aR expression and suggest a maturation-related change in signaling of the C5aR, presumably at the level of receptor coupling.
EDTA-blood samples derived either from healthy staff or septic patients were investigated for in vitro complement activation during the first 48 h after blood drawing at 4 degrees C and 20 degrees C. For this purpose C3a/C3a desArg plasma levels were determined by the ABICAP C3a assay. Within the septic group no complement activation was detectable during the whole observation period. However, if blood from healthy persons was stored for longer than 6 h at 20 degrees C complement activation occurred. The most profound activation was found in EDTA-blood stored for 48 h at 20 degrees C. C3a values in this sample increased four-fold from 56 +/- 7 ng/ml to 222 +/- 38 ng/ml. From these data we conclude that both immediate cooling of EDTA-blood to 4 degrees C, as well as the immediate separation of plasma as proposed by Mollnes et al. (Clin. Exp. Immunol. (1988) 73, 484), is not necessary for determination of anaphylatoxin plasma values. Storage of EDTA-blood samples for up to 6 h without the need to perform centrifugation should allow anaphylatoxin measurement to become a routine parameter for diagnosis of inflammatory diseases.
The anaphylatoxic peptide C5a is an important inflammatory mediator of the complement system. We have generated human C5a-receptor (hC5aR) mutants with truncation of its cytosolic carboxyl-terminus (C-terminus). Both mutants were analysed for C5a-binding in transiently expressing COS cells, and one mutant additionally for GTP-binding regulatory protein (G-protein) coupling in cRNA-injected Xenopus oocytes. Our data suggest that (a) amino acids (aa) 314 to 326 as part of the C-terminus are necessary for proper receptor folding or expression and (b) the receptor C-terminus distal from position 327 is not critical for receptor expression, folding, binding and G-protein coupling.
To check and clarify existing data on receptor-interacting residues in the human C5a anaphylatoxin, we tested mutant C5a proteins obtained by site-directed mutagenesis of a recombinant human C5a (rhC5a) cDNA clone for structural and functional integrity. Amino acid positions in three different regions of the molecule were investigated: Arg74 at the C-terminus, Arg40 and Pro45 located in the core region, and Lys14 and Lys19, Lys20 in the N-terminus. Des-Arg74-rhC5a displayed only a residual 3-4% functional activity in the myeloperoxidase-release assay from human granulocytes while retaining the three-dimensional solution structure of wild-type (wt)-rhC5a as shown by circular dichroism (CD) spectroscopy. Des-Arg74-rhC5a was able to activate the human C5a receptor transiently expressed in Xenopus oocytes, but was inactive in the heterologous guinea pig (gp) ileum-contraction assay. These results reveal profound differences between the guinea pig and human C5a-receptor ligand-binding characteristics. Exchange of the core residue Arg40 by a glycine did not significantly affect functional C5a activity, in contrast to a previous observation [Mollison, K. W., Mandecki, W., Zuiderweg, E. P., Fayer, L., Fey, T. A., Krause, R. A., Conway, R. G., Miller, L., Edalji, R. P., Shallcross, M. A., Lane, B., Fox, J. L., Greer, J. & Carter, G. W. (1989) Identification of receptor-interacting residues in the inflammatory complement protein C5a by site-directed mutagenesis, Proc. Natl Acad. Sci. USA 86, 292-296], nor did exchange of the conserved Pro45 residue by the C3a analogue glutamic acid, a mutation expected to alter the whole geometry of the loop connecting helix III-helix IV (including Arg40) of the C5a molecule. Thus, participation of this loop in receptor interaction appears unlikely. While exchange of the N-terminal Lys14 residue by alanine did not significantly affect functional activity, a double replacement of Lys19 and Lys20 by alanine residues reduced activity more than 30-fold. These results confirm Lys19 and/or Lys20 as a putative receptor-interacting site, although we could not obtain a CD spectrum of this important mutant due to poor expression.
The colonization of hospitalized patients by Pseudomonas aeruginosa at an intensive care unit for burn victims was studied over a one-year period. A total of 231 isolates from 12 patients were analyzed by macrorestriction analysis. The results revealed that most patients were infected with only one to three different strains. In several patients Pseudomonas aeruginosa isolates of the same clonal lineage exhibited considerable differences in their macrorestriction fragment pattern. Digestion with further restriction enzymes, however, allowed differentiation between clonal variants due to a high genetic drift and superinfection with a different Pseudomonas aeruginosa strain. Isolates of the same clonal lineage could be isolated from several patients as well as from the patients' environment. Notably, Pseudomonas aeruginosa could be isolated from sedimentation plates. Thus, patients may have been extensively cross-infected on the ward. These data underline the importance of strict infection control measures and of regular surveillance for Pseudomonas aeruginosa by an appropriate typing method, i.e. one that can differentiate strains with high genomic variability.