Molecular Motions and Interactions in Aqueous Solutions of Thymosin-ß4 , Stabilin CTD and Their 1 : 1 Complex, Studied by 1 H-NMR Spectroscopy.

Wide-line 1 H NMR measurements were extended and all results were interpreted in a thermodynamics-based new approach on aqueous solutions of thymosin-ß4 (Tß4 ), stabilin cytoplasmic domain (CTD), and their 1 : 1 complex. Energy distributions of potential barriers controlling the motion of protein-bound water molecules were determined. Heterogeneous and homogeneous regions were found in the protein-water interface. The measure of heterogeneity of this interface gives quantitative value for the portion of disordered parts in the protein. Ordered structural elements were found extending up to ∼20 % of the individual whole proteins. About 40 % of the binding sites of free Tß4 get involved in bonds holding the complex together. The complex has the most heterogeneous solvent accessible surface (SAS) in terms of protein-water interactions. The complex is more disordered than Tß4 or stabilin CTD. The greater SAS area of the complex is interpreted as a clear sign of its open structure.

Comparison of hypotensive response to aggregated IgG or to bacterial LPS in rats.

OBJECTIVE AND DESIGN: Rats treated with aggregated IgG (Aggr.) become "refractory" to the hypotensive action of a second dose of Aggr. The objective of this study was to assess the responsiveness of animals pretreated with Aggr. to bacterial LPS and vice versa. MATERIAL OR SUBJECTS: Female Wistar rats (250-300 g) were used. Each experiment was carried on at least 4 animals. TREATMENT: A human IgG preparation containing 30% aggregates (10-16 mg/100 g) or E. coli serotype 0111.B4 (0.005-mg/100 g) was administered i.v. Certain groups of animals were pretreated with 1 mg/100 g GdCl3 or with 10 mg/100 g pentoxyphylline (PTX). METHODS: Arterial blood pressure was monitored in the carotis-using a polyethylene cannula and an electronic tension meter. Tumor necrosis factor alpha (TNF-alpha) activity was estimated by the use of an L-929 cell cytotoxicity assay. RESULTS: Pretreatment of rats with a sublethal dose of LPS impaired the hypotensive reaction of the animals to Aggr. Rats male "refractory" to Aggr. reacted to the injection of LPS with hypotension and a second phase milder than in the controls. Hypotension could not be elicited by Aggr. in rats pretreated with GdCl3. The same pretreatment had no effect on the first phase of hypotension induced by intravenous injection of LPS, whilst a mitigation of the second phase was observed. Infusion of PTX immediately prior to Aggr. administration prevented the drop of blood pressure. A sizeable level of TNF-alpha was detected only later than blood pressure had reached its minimum level following Aggr. administration. CONCLUSIONS: Hypotension induced by LPS may involve a macrophage population broader than that responsible for the vascular action of Aggr. The data presented do not support a primary role for TNF-alpha in Aggr. induced hypotension.

Hypotensive action of aggregated IgG in rats. Study of the unresponsiveness to a second dose following restoration of the blood pressure.

Human IgG preparations containing aggregates have been reported to induce hypotension in rats. Animals surviving a hypotensive dose of such a preparation were found unresponsive to a second dose given the next day. Unresponsiveness was induced by an isolated fraction of aggregated IgG, but not by purified monomers. Preparations containing aggregated and monomeric IgG appeared more efficient in inducing unresponsiveness than aggregates alone. The phagocyte function in vivo was assessed in unresponsive animals. No significant difference was found in the carbon clearance and in the human erythrocyte clearance test suggesting that a general impairment of the phagocyte function is not necessary for unresponsiveness. Previous studies indicated the involvement of PAF in hypotension by aggregated IgG. Hypotension was elicited by the injection of PAF in rats made "refractory" to aggregated IgG like in controls.

Hypotensive action of IgG preparations containing aggregates is suppressed by PAF-receptor antagonist BN 52021 and by gadolinium chloride (an agent blocking Kupffer cell function).

The hypotensive side effect of four IgG preparations was studied in a rat model. An unmodified, chromatographically isolated preparation (prepn. C) appeared to induce a transient drop in blood pressure via the kininogen kinin system. A preparation made by ethanol fractionation (prepn. E) and an ethanol-fractionated, modified product (prepn. M) produced an action which does not depend on kininogen activation. No blocking of kininase was required for this effect and it was brought about by prepn. M was devoid of any sizeable prekallikrein activator or kallikrein like proteinase content. The effect was associated by the aggregate fraction of prepn. E, but not with IgG monomers of the same preparation. Animals given these latter types of IgG preparations were refractory to a second dose 24 hours later. The involvement of PAF in the kinin-independent hypotensive action is suggested by the finding that PAF-receptor antagonist BN 52021 prevented the effect and returned the blood pressure close to normality when given during hypotension. A blockade of Kupffer cells brought about by administration of GdCl3 was also found to prevent the effect pointing to the involvement of these type of cells in the reactions.