Nigel D. Staite

Acute and chronic inflammatory responses to local administration of recombinant IL-1α, IL-β, TNFα, IL-2 and Ifnγ in mice

The contention that cytokines are important mediators of inflammation prompted the present studies which were designed to compare acute and chronic pathological effects of locally-administered recombinant (r) IL-1α, IL-1β, TNFα, IL-2 and Ifnγ. Acute (6 hr), resolving (48 hr) inflammation was induced by the following, in order of potency: rIL-1α>rIL-1β>rTNFα>rIfnγ=BSA (control) following a single sc. injection. However, only rIL-1β and rIL-2 initiated and maintained chronic granulomatous reactions when delivered locally from a sc. ethylene vinyl acetate (EVA) slow-release polymer. The predominance of macrophages in EVA-rIL-1β lesions contrasted with the proliferative lymphoid granulomata induced by EVA-rIL-2 implants. These “in vivo” observations reinforce, the roles of both IL-1β and IL-2 as potent mediators of chronic immunoinflammatory disease.

Anti-inflammatory/antiarthritic ketonic bisphosphonic acid esters

Bisphosphonate ester 2 is an inhibitor of inflammation, but is devoid of antiarthritic effects. SAR studies on a series of related bisphosphonate esters resulted in compounds 6e, 6i, 6j, and 6m, which exhibited excellent inhibition of an arthritis model, in addition to potent anti-inflammatory effects.

New Anti-Inflammatory/Anti-Arthritic Heterocyclic Bisphosphonates

Abstract. In the course of research toward a safe and effective treatment for rheumatoid arthritis, we identified new pyrazolo[1,5-a]pyrimidine and 4-pyrimidinone bisphosphonate esters, which are potent inhibitors of a murine model of chronic, cutaneous inflammation (delayed type hypersensitivity granuloma) and a murine antigen induced arthritis model. 9a has EC30 values of 0.01 and 0.005 mg/kg respectively and represents a new class of antiinflammatory/antiarthritic bisphosphonate ester.

Chemical modification of Interleukin-1β: Biochemical characterization of a carbodiimide-catalyzed intramolecular cross-linked protein

We have modified recombinant human Interleukin-1β using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide atpH 6.5, resulting in the formation of an internally cross-linked protein. The major product (30% yield) of the reaction (17 kD; pI=6.2) was purified and fully characterized by peptide mapping using Endoproteinase Lys C. When digests were conducted under nondenaturing conditions, we found that the modified protein is different from the native protein. The native protein yielded 14 peptides after digestion, whereas only two large peptides and a tetrapeptide, Asn-Tyr-Pro-Lys, were released from the cross-linked protein (i.e., cleavage occurs only at residues Lys88 and Lys92). Using gel filtration, the two peptides were found to co-elute as a single species (15 kD), which represent a noncovalent complex of the amino terminal and C-terminal portions of the molecule. Further analysis of the modified protein by peptide mapping under denaturing conditions and by FAB MS analysis showed that Glu111 and Lys138 were internally cross-linked. The cross-linked protein had bioactivity (T-cell proliferation), fluorescence, and circular dichroism spectra similar to native IL-1β. In contrast, while having similar secondary structure, the digested cross-linked protein had less than 1% of T-cell proliferative activity of the undigested protein. These data show that the structural integrity surrounding and perhaps including the Asn-Tyr-Pro-Lys region may be crucial for the biological activity of rIL-1β and may be important for the binding of IL-1 to its receptor.

Isolation and bioactivities of three IL-1β N-terminal variants

Three distinct N-terminal variants of rhIL-1β can be generated by expression of the IL-1β gene in E. coli; the naturally occurring Ala1 species, Met0-Ala1 and des-Ala1 proteins. Since most studies with rhIL-1β have used a mixture of two or more variants, we have evaluated their individual bioactivities. The variants were resolved by cation exchange HPLC. Bioactivity measurement on murine thymocytes gave a potency order of Ala1 > des-Ala1 > Met0-IL-1β. Analysis using human T-cells co-stimulated with PMA showed a potency order of Ala1 > des-Ala1 > Met0-IL-1β. Thus changes in the N-terminal amino acid of IL-1β changes the activity of the protein. Since murine and human T-cells respond similarly, the interactions between the N-terminus of rhIL-1β and their receptors probably occur through comparable mechanisms.