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To check and clarify existing data on receptor-interacting residues in the human C5a anaphylatoxin, we tested mutant C5 a 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 Lysl4 and Lysl9, 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) -rhC5 a as shown by circular dichroism (CD) spectroscopy. Des-Arg74-rhC5a was able to activate the human C5 a 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 C5 a by sitedirected mutagenesis, Proc. Nutl Acad. Sci. USA 86, 292-2961, 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 I11 -helix IV (including Arg40) of the C5a molecule. Thus, participation of this loop in receptor interaction appears unlikely. While exchange of the N-terminal Lysl4 residue by alanine did not significantly affect functional activity, a double replacement of Lysl9 and Lys20 by alanine residues reduced activity more than 30-fold. These results c o n f m Lysl9 a n d 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 human C5a anaphylatoxin is a small protein of 74 amino acids which mediates a variety of cellular activation and release processes. It is generated at sites of inflammation with concomitant complement-system activation by proteolytic cleavage from the a chain of the complement factor C5. The major target cells are the granulocyte and macrophage, leading to chemotactic attraction of neutrophils, induction of the oxidative burst and release of lysosomal enzymes, histamine and interleukins. All responses are mediated by interaction with the membrane-bound C5a receptor, which has recently been cloned (for review, see Kohl and Bitter-Suermann, 1993).Identification of receptor-interacting residues in the human C5 a molecule are essential for the rational development of receptor antagonists which are of considerable therapeutic

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Escherichia coli O157 lipopolysaccharide (LPS) was developed with sera from 63 children with confirmed recent E. coli O157 infection and from 256 age-stratified urban controls. The median ELISA values for control and case sera were 0.05 (interquartile range, 0 to 0.20; mean ؎ standard deviation [SD], 0.15 ؎ 0.22) and 1.41 (interquartile range, 1.11 to 1.59; mean ؎ SD, 1.41 ؎ 0.53), respectively (P < 0.001). With a breakpoint of 0.59 (mean ELISA value of the control sera ؉ 2 SDs), the assay had a sensitivity, specificity, and positive and negative predictive values of 95, 94, 80, and 98%, respectively, for recent E. coli O157 infection. The O157 LPS assay and Vero cytotoxin (VT) 1-neutralizing-antibody (NAb) assay were used to compare the relative frequencies of O157 LPS antibodies and VT1-NAbs in an age-stratified urban population from Toronto, Ontario, Canada, and in 216 healthy family members from dairy farms in southern Ontario. The frequency of O157 LPS antibodies was about threefold higher in dairy farm residents (12.5%) than in urban residents (4.7%) (P < 0.01). Similarly, the frequency of VT1-NAbs was about sixfold higher in dairy farm residents (42.0%) than in urban residents (7.7%) (P < 0.001). These findings are consistent with a greater level of exposure of dairy farm residents to VT-producing E. coli (VTEC) strains. The high rate of seropositivity to VT1 in farm residents probably reflects the booster effect of repeated VTEC exposures and argues against a sustained generalized immunosuppressive effect of VT1. Seroepidemiological studies may help in assessing the level of exposure of different populations to VTEC strains.

Alveolar type II (AT2) epithelial cells have important functions including the production of surfactant and regeneration of lost alveolar type I epithelial cells. The ability of in vitro production of AT2 cells would offer new therapeutic options in treating pulmonary injuries and disorders including genetically based surfactant deficiencies. Aiming at the generation of AT2-like cells, the differentiation of murine embryonic stem cells (mESCs) toward mesendodermal progenitors (MEPs) was optimized using a "Brachyury-eGFP-knock in" mESC line. eGFP expression demonstrated generation of up to 65% MEPs at day 4 after formation of embryoid bodies (EBs) under serum-free conditions. Plated EBs were further differentiated into AT2-like cells for a total of 25 days in serum-free media resulting in the expression of endodermal marker genes (FoxA2, Sox17, TTR, TTF-1) and of markers for distal lung epithelium (surfactant proteins (SP-) A, B, C, and D, CCSP, aquaporin 5). Notably, expression of SP-C as the only known AT2 cell specific marker could be detected after serum-induction as well as under serum-free conditions. Cytoplasmic localization of SP-C was demonstrated by confocal microscopy. The presence of AT2-like cells was confirmed by electron microscopy providing evidence for polarized cells with apical microvilli and lamellar body-like structures. Our results demonstrate the differentiation of AT2-like cells from mESCs after serum-induction and under serum-free conditions. The established serum-free differentiation protocol will facilitate the identification of key differentiation factors leading to a more specific and effective generation of AT2-like cells from ESCs.