Frequency of human anti-FVIII antibodies in humanized SCID mice elicited by recombinant deleted factor VIII and by a plasma derived factor VIII.

SCID mice were grafted with human PBL (hu-PBL-SCID) from healthy or haemophilia A donors. Those containing human and no murine Ig in their plasma, were injected with 100 U VIII:Ag of a plasma derived (pd) FVIII or recombinant deleted Factor VIII (FVIII deltaII) and with 10 microg of tetanus toxoid as control immunogen. The frequency and the intensity of the humoral specific responses were measured in 253 mice humanized with PBL from 13 different donors. There was no significant difference in the frequency or intensity of the anti-FVIII immune responses to pd FVIII and FVIII deltaII. Neutralizing antibodies were only detected in the plasma of mice humanized with cells from haemophiliacs having FVIII inhibitors in their blood. The immune responses observed in hu-PBL-SCID mice correlated with the immune status of the corresponding human donor.

Liquid pasteurization of an immunoglobulin preparation without stabilizer: effects on its biological and biochemical properties.

Intravenous immunoglobulins (IVIg) purified by cold ethanol fractionation have a very good safety record with regard to the transmission of many viruses. However, a few cases of non-A-non-B hepatitis have been described after intravenous injection of some immunoglobulin preparations. To ensure even higher safety for our IVIg, an additional virus inactivation step, based on pasteurization, was developed. The heating of aqueous IVIg was performed without stabilizer, and at a very low salt concentration (< 1 mM) at acidic pH. No generation of polymer was detected after pasteurization and a significant decrease in the proportion of dimers was observed. Analysis of the secondary structure by circular dichroism showed a very slight change in the secondary structure. The biological properties of the Fc region as well as the Fab region were not affected by the pasteurization. Our method has several advantages: (1) improvement of viral safety; (2) there is no need to add stabilizer which may stabilize viral particles, and (3) the absence of any hypotensive effect and low anticomplementary activity indicates a good clinical tolerance of IgG preparation.

Characterization of one alpha 2-macroglobulin with anticomplementary activity from pregnant women.

In 15 pregnant women during the third term of pregnancy, the immunomodulatory property of alpha 2-macroglobulin (alpha 2M) was initially detected by measuring the inhibitory effect on immune complement-dependent haemolysis of serum alpha 2M fractions obtained by gel filtration. By a two-step chromatography procedure consisting of gel filtration followed by anion-exchange chromatography, different sub-forms of alpha 2M in serum were separated. Amongst them, it was shown that the inhibition of complement activity was almost exclusively linked to one particular subform. Additional studies revealed that the observed effect was not due to proteases bound to alpha 2M during clotting since, by using protease-specific inhibitors, no change was observed in complement inhibition. This subform, though present at very low levels in control sera, appeared in strikingly increased amounts during the third trimester of pregnancy (35 mg/l) and comprised between 3 and 5% of the total alpha 2M. Results show that the increase of alpha 2M anticomplementary activity is linked to the increase in alpha 2M levels in serum.

The phagocytic activity of Lumbricus terrestris leukocytes is enhanced by the vertebrate opsonins: IgG and complement C3b fragment.

The effect of the vertebrates opsonins: IgG and complement C3 fragments on phagocytic activity of Lumbricus terrestris leukocytes towards sheep erythrocytes was studied. Sheep erythrocytes were previously sensitized with specific IgG or IgM and coated with one of the third component fragments (C3b, C3bi, C3d) of human complement. Our results show that leukocyte phagocytosis was enhanced by vertebrate IgG and C3b complement fragments but not by IgM and fragment C3d. Because opsonization in vertebrates is related to the presence of receptors on the surface of phagocyte membrane, our results suggest that similar receptors exist on earthworm leukocyte surfaces. These new data strengthen the arguments in favour of the presence of components in Lumbricus terrestris which partially share common structures and functions with components of vertebrate humoral immune reactions.

Evidence of synthesis by Lumbricus terrestris of specific substances in response to an immunization with a synthetic hapten.

Lumbricus terrestris (Annelid, Oligocheta) is capable of cellular- and humoral-specific reactions against natural antigens. Is this earthworm able to elaborate a response of antibody type against a synthetic hapten? L. terrestris have been immunized with conjugates made of one of two different synthetic haptens (400 mw) and a carrier protein: bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH). The presence of anti-hapten substances in coelomic fluid was tested against each iodinated hapten derivative (125I-hapten). The 125I-hapten-substance complexes were separated from the free derivatives by polyethylene glycol (PEG) 6000 precipitation or by gel filtration. The experiments showed that L. terrestris synthesized specific substances against the immunizing hapten. The importance of the response depended on the carrier protein and on the amount of introduced immunogen. A kinetic study of first and second immunization showed that these substances, elaborated in response to the immunization with the synthetic hapten, were synthesized by cells which kept the immunological memory. These data are discussed in relation to the humoral protection mechanisms already established in the invertebrates.

[Substances contained in the coelomic fluid of Lumbricus terristris having functions in common with those of some human complement components]. / Mise en évidence de substances contenues dans le liquide coelomique de Lumbricus terrestris, possédant des fonctions communes avec celles de certains composants du complément humain.

In the present work, it was demonstrated for the first time that the coelomic fluid of Lumbricus terrestris contained a substance recognized by human classical convertase. This substance permitted an immune, complement-dependent agglutination response of sheep erythrocytes carrying C3-convertase (EAC142). In addition, substances secreted by coelomic leukocytes possessed an inhibitory activity against human complement: the component C3 was cleaved into a C3b fragment. This result suggests the presence of a system the function of which is C3-convertase-like. We have thus shown that some complement functions, with their natural inhibitors, appear early in evolution. Certain of these functions and structures might be preserved throughout evolution.