Alexander Steinkasserer

Polymorphism in human IL-1 receptor antagonist gene intron 2 is caused by variable numbers of an 86-bp tandem repeat

We have investigated the polymorphism in intron 2 of the interleukin-1 receptor antagonist gene and identified two new alleles of the system. We have shown that the polymorphism is caused by the variable copy number of an 86-bp sequence, by using the polymerase chain reaction and primers immediately flanking the repeat region, and by direct sequencing. The repeat region contains three potential protein-binding sites and therefore the variable copy number may have functional significance.

Human β2-glycoprotein I: molecular analysis of DNA and amino acid polymorphism

In this study, we describe a two-allelic RsaI restriction fragment length polymorphism identified by Southern blot analysis and by allele-specific polymerase chain reaction amplification for the human β2-glycoprotein I (β2-I; apolipoprotein H=APOH) gene. This polymorphism, which segregates in a co-dominant fashion, leads to a valine-leucine amino acid exchange at amino acid position 247. The allele frequency has been established in 34 unrelated parents of the Centre dEtude du Polymorphisme Humain family panel and was found to be 0.76 for valine and 0.23 for leucine. The Val-Leu polymorphism described in this study does not correlate with the four isoelectric focusing alleles previously described, indicating that other variants are responsible for this polymorphism.

Use of the acute phase serum amyloid A2 (SAA2) gene promoter in the analysis of pro- and anti-inflammatory mediators: differential kinetics of SAA2 promoter induction by IL-1β and TNF-α compared to IL-6

A cytokine responsive construct, pGL2-SAA2pt, was generated by cloning the acute phase promoter of human serum amyloid A2 (SAA2) upstream of a luciferase reporter gene. The construct responds to the inflammatory mediators MoCM, IL-1 beta, TNF-alpha, and IL-6 in a manner that closely mimics the response of the endogenous SAA2 gene to such stimuli: i.e. single treatments induce transcriptional activation by IL-1 beta and TNF-alpha to a greater extent than by IL-6 at 12-24 h. However, timecourse experiments show that the kinetics of induction generated by IL-1 beta and TNF-alpha are quite distinct from IL-6, IL-6 having a much greater effect at 3-6 h. IL-1 beta and TNF-alpha synergize with IL-6 to give a 10-fold increase in transcriptional readout over single cytokine treatments. The kinetics of this synergistic response resembles that generated by IL-6 alone. The IL-1 receptor antagonist, hIL-1ra, can specifically block the IL-1 beta driven transcriptional activation of pGL2-SAA2pt, but not that driven by TNF-alpha or IL-6. Furthermore, in synergistic cytokine combinations, it blocks only the IL-1 beta driven component indicating that the effect is biological and not attributable to toxicity. Consequently assays utilizing pGL2-SAA2pt will be useful both for the investigation of the kinetics of inflammatory signalling in a cytokine specific manner, and for the evaluation of the pro- and anti-inflammatory properties of novel natural and synthetic molecules.

Human Interleukin‐1 receptor antagonist High yield expression in E. coli and examination of cysteine residues

The human IL‐1 receptor antagonist (IL‐1ra) was produced in a high yield E. coli expression system, and was purified in a rapid two‐step purification, This recombinant IL‐1ra molecule possessed full binding activity to the IL‐1 receptor (type I) and totally inhibited IL‐1‐induced PGE2 production by human dermal fibroblasts. Radioalkylation and analysis of V8‐derived IL‐1ra peptides indicate that the four cysteines present in the IL‐1ra are not disulphide‐linked.

Human interleukin-1 receptor antagonist is expressed in liver

Using PCR and Northern blot analysis, an IL‐1 receptor antagonist specific transcript was amplified from HepG2‐ and liver mRNA, cDNA clones coding for IL‐1 receptor antagonist were isolated from a liver cDNA library and sequence comparison revealed complete identity with the secreted, monocytic form of IL‐1 receptor antagonist.

Expression of human recombinant β2‐glycoprotein I with anticardiolipin antibody cofactor activity

To enable the synthesis of β2‐glycoprotein I mutants we have established a stable Chinese hamster ovary cell line that expresses human β2‐glycoprotein I up to 2.9 μg/106 cells/day. Recombinant β2‐glycoprotein I is identical to the purified native protein with respect to cofactor activity revealed in a modified anti‐cardiolipin ELISA. Autoimmune type anti‐cardiolipin antibody requires recombinant β2‐glycoprotein I in a dose‐dependent manner to bind cardiolipin whilst binding of infectious type antibody is inhibited. The purified recombinant β2‐glycoprotein I in serum free medium exists as two oligosaccharide species which upon deglycosylation have identical apparent molecular weight to the deglycosylated native protein.