No description available.

No description available.

The complement system is one of the most ancient immune defense mechanisms, providing rapid protection against invading micro-organisms. It is essential for the complement cascade to be under tight regulation in order to prevent excessive production of complement proteins. C5a is the most potent anaphylotoxin produced by the complement system, it binds C5a receptor (C5aR, CD88) and C5L2 (GPR77). C5a binding to C5aR induces leukocyte chemotaxis and release of inflammatory mediators. Over-production of C5a is known to be involved in many inflammatory and pathological conditions such as RA, I1R injury and sepsis, making it an attractive therapeutic target. Human and mouse C5aR share low homology and blocking C5a/C5aR signaling with small molecules has been challenging. We generated human C5aR knockout/knockin (hC5aR KI) mice in which the mouse C5aR coding region was replaced with that of human C5aR to utilize them for the development of human therapeutics targeting C5aR. hC5aR KI mice showed normal development, and leukocytes from hC5aR KI mice responded well to mouse C5a. We used two approaches to generate monoclonal antibodies (mAbs) against hC5aR. We used a mouse cell line transfected with hC5aR or neutrophils from hC5aR KI mice to immunize wild-type mice and generated high-affinity antagonistic mAbs which are specific to human C5aR. Anti-hC5aR mAb 7F3 blocked C5a-induced signaling completely without agonistic activity in vitro. In the animal model of K/BxN inflammatory arthritis, 7F3 both prevented and reversed inflammation. Currently, the function of the second C5a receptor, C5L2, remains controversial. There are contradicting reports from C5L2 KO mice that were generated by independent groups. We assessed the function of human C5L2 using an antagonistic mAb that specifically blocks C5L2 function and not C5aR. In vitro analysis using the C5L2-blocking mAb showed that C5a does not signal via C5L2 to affect chemotaxis or phagocytosis by neutrophils, indicating that C5L2 is not a signaling receptor for C5a, at least in these cellular functions.

1. A method for the purification of the serine protease,factor D,was developed using conventional chromatographic procedures. The final product was homogeneous as judged by SDS/polyacrylamide gel electrophoresis, its migration as a single component in ion exchange and gel filtration media, and its amino acid sequence analysis. The molecule had an apparent molecular weiyht of 24,000. It contained <1.5% (w/w) reducing sugars as judged by periodic acid/Schiff staining, and existed as a monomer in buffers containing either EDTA or calcium ions. 2. Approximately 84% of the amino acid sequence was established unequivocally by automated sequence analysis of the intact molecule and peptides derived by digestion with CNBr, o-iodosobenzoic acid, trypsin and V8 protease. Carboxypeptidase-Y digestion was used to establish the C-terminal amino acid. The peptides were aligned either by homology with other serine proteases, or by the overlap of sequences obtained from peptides derived by different fragmentation procedures. The molecule nad a typical serine protease-type sequence with isoleucine as the Nterminal amino acid. The active site serine and aspartic acid and the surrounding sequences were conserved as well as the sequence around the position of the active site histidine, although this residue itself was not identified. 3. The possibility of the existence of a factor D̄ zymogen which can be activated by trypsin was reinvestigated, but no evidence for a precursor was found. No enzymic activity towards a number of p-nitroanilide substrates and arginyl and lysyl esters was observed with factor D̄,but it was found to release p-nitrophenol from p-nitrophenyl-p'-guanidinobenzoate. Factor D̄ was inhibited by diisopropylphosphofluoridate and p-nitrophenyl-p'-guanidinobenzoate, but a variety of other non-protein and protein inhibitors including α₂-macroglobulin, c1 inhibitor and inter-α-trypsin inhibitor had no effect on enzymic activity.

Thesis (M.Sc.) -- University of Adelaide, Dept. of Microbiology and Immunology, 1983. / Typescript (photocopy).

No description available.

1. The DNA sequence of the human complement C4A gene from a cosmid clone Cos 3A3 was determined and the complete exon-intron structure elucidated. The 5' flanking region of the C4 gene contains three TATA sequences and a transcriptional enhancer core sequence, which are >200 nucleotides (nt) and 60-70 nt upstream from the CAP site, respectively. The gene consists of 42 exons coding for a precursor protein of 1745 residues. The first exon codes for a 51 nt 5' untranslated sequence, a leader peptide of 19 residues, and the N-terminus of the β chain. The β-α and the α-γ chain junctions are encoded by exons 17 and 34, respectively. The anaphylatoxin C4a and the thiolester site are encoded by phase 1-1 symmetrical exons. Most of the amino acids encoded at the splice junctions are polar or charged. Between exons 10 and 11 is a 6-7 kb intron that is flanked by direct long terminal repeats and may be absent in some C4 genes located at the second C4 locus. The last exon codes for the C-terminus of the γ chain and a 140 bp 3' untranslated sequence. The intergenic region between the C4 gene and its neighbouring 21-hydroxylase (210Hase) gene is ~3028 bp. 2. Eighteen polymorphic amino acids on C4 have been identified through genomic DNA, cDNA and protein sequencing. Fourteen of them are located on the* chain (C4a: 2 changes; C4d: 12 changes). The rest are scattered on the β and the γ chains. There are potential size variations by one residue on the β chain, and by a tripeptide that contains a sulphation site on the α chain. 3. Four common and rare C4 alleles have been cloned from individuals whose C4 proteins were chemically and serologically characterised. Analysis of the sequences at the C4d regions has allowed the identification of the C4A/C4B isotypic residues at positions 1101-6: C4A has the sequence PCPVLD, while C4B has the sequence LSPVIH. Presumably these isotypic residues are the cause of the class-specific, differential chemical reactivates. Moreover, the probable locations for the two Eodgers (Kg) and the six Chido (Ch) antigenic determinants were deduced. The C4B isotypic residues may be involved in the expression of the Ch2 and the Ch4 epitopes, while the C4A isotypic residues may not be related to either of the Eg determinants. 4. Definitive restriction fragment length polymorphisms (RFLPs) representing the exact locations responsible for the isotypicity between C4A and C4B, and for their generally associated Rg1 and Ch1 antigenic determinants, have been designed. In combination with the Taq I polymorphic patterns specific for the C4 and for the 210Hase gene loci, it has been shown that the null allele of the HLA haplotype B44 DR6 C4A 3 C4B QO is not a C4B allele, but probably encodes another C4A 3 allotype at the second C4 locus.

Typescript (photocopy) / viii, 102, xxv leaves, [6] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.) Dept. of Microbiology, University of Adelaide, 1982

Thesis (Ph.D.) Dept. of Microbiology, University of Adelaide, 1982. / Typescript (photocopy).

This work attempts to establish some phenomenon of a rise in titer of C'3 and C'4 due to antigenic stimulation. Normal level of complement is established and compared to other workers as well as against guinea pig levels. Young rats were bled to establish normal levels of complement. The animals were then injected with an antigenic substance and after a period of seven days were bled again to determine the level of complement. Various antigenic and non-antigenic substances were used as well as normal saline injections for control.