Amanda J. Bradley

Biophysical consequences of linker chemistry and polymer size on stealth erythrocytes: size does matter.

Immunocamouflaged red blood cells (RBC) are produced by cell surface derivatization with methoxypolyethylene glycol (mPEG). These immunologically attenuated cells may reduce the risk of allosensitization in chronically transfused patients. To characterize the effects of differing linker chemistries and polymer lengths, RBC were modified with cyanuric chloride activated mPEG (C-mPEG 5 kDa), benzotriazole carbonate methoxyPEG (BTC-mPEG; 5 or 20 kDa) or N-hydroxysuccinimidyl ester of mPEG propionic acid (SPA-mPEG; 2, 5 or 20 kDa). Biophysical methods including particle electrophoresis and aqueous two-phase polymer partitioning were employed to compare the PEG derivatives. While C-mPEG was faster reacting, both BTC-mPEG and SPA-mPEG gave comparable findings after 1 h. Both PEG surface density and molecular mass had a large effect on RBC surface properties. Proportional changes in electrophoretic mobility and preferential phase partitioning were achieved by increasing either the quantity of surface PEG or the PEG molecular mass. In addition, two-phase partitioning may provide a means for efficiently removing unmodified or lightly modified (hence potentially immunogenic) RBC in the clinical setting. Furthermore, mPEG modification significantly inhibits cell-cell interaction as evidenced by loss of Rouleaux formation and, consequently, sedimentation rate. Importantly, BTC-mPEG 20 kDa RBC showed normal in vivo survival in mice at immunoprotective concentrations (up to 2 mM).

The complement system in liposome clearance : Can complement deposition be inhibited?

The activation of complement results in the opsonization of particles for removal by the reticuloendothelial system. Experimental evidence suggests that complement-mediated clearance of liposomal systems may significantly contribute to liposome biodistribution. Because of the multiplicity of complement activation mechanisms and the large number of proteins in the pathway, there are multiple opportunities to reduce or eliminate the opsonic effects of complement activation. This review addresses the state of our understanding of the interaction of liposomes with complement proteins and suggests some approaches to minimize complement activation.

Interactions of IgM ABO antibodies and complement with methoxy-PEG-modified human RBCs

BACKGROUND: RBCs modified with cyanuric chloride activated methoxy‐PEG (CmPEG; 5000 Da) are less immunogenic than untreated RBCs, and their use thus may reduce the risk of alloimmunization in chronically transfused patients.

Analysis of immunoglobulin class, IgG subclass and titre of HPA‐1a antibodies in alloimmunized mothers giving birth to babies with or without neonatal alloimmune thrombocytopenia

We analysed the titre and isotype composition of antibodies produced by mothers giving birth to babies with or without neonatal alloimmune thrombocytopenic purpura (NAITP) and patients with post‐transfusion purpura (PTP). All these individuals produced an antibody specific for the HPA‐1a allotype present on the platelet glycoprotein IIb‐IIIa (GPIIb‐IIIa). Sera from mothers who gave birth to thrombocytopenic babies (group 1, n = 36), non‐thrombo‐cytopenic babies (group 2, n = 4) or from PTP patients (group 3, n = 3) were tested by an indirect‐ELISA. Results indicated no evident differences in the isotype composition or titre of the antibodies from the three groups of sera. The antibody titre ranged from 1: 120 to 1: 3500. Antibodies with the IgGl subclass were present in all sera. Most sera contained IgGl alone (24/43 sera tested) or in combination with IgG3 (10/43). IgG2 was never present and only three sera showed intermediate reactivity with anti‐IgG4 MAb. Few sera (nine sera from groups 1 and 2) were weakly positive when tested with the anti‐IgM antibodies. These results suggest that neither the titre nor the isotype composition can be used to predict the severity or the occurrence of thrombocytopenia in newborns.

Camouflaged blood cells: Low-technology bioengineering for transfusion medicine?

The small number of studies done on the covalent modification of RBC with PEG, or PEG-derivatives, suggests that the immunocamouflage of intact cells significantly reduces the antigenicity and immunogenicity of the foreign cell. Importantly, this protective immunologic effect can be accomplished without adversely affecting the structure, function, or viability of the modified cell (e.g., RBCs and lymphocytes). As a consequence, PEG-RBC may have significant practical value in the treatment of the chronically transfused patient as a prophylactic measure against allosensitization. The PEG-RBC also may be useful in treating the already allosensitized individual. As shown, preexisting antibodies do not effectively recognize nor bind to the modified donor cells. A finding of further interest to transfusion medicine is that pegylation of contaminating lymphocytes within RBC products may prove efficacious in preventing graft-versus-host disease in the immunocompromised patient. However, the main emphasis of our research continues to be the immunocamouflage of RBC for use in chronic transfusion therapy of the SCD and thalassemic patient.

C1q Binding to liposomes is surface charge dependent and is inhibited by peptides consisting of residues 14–26 of the human C1qA chain in a sequence independent manner

Complement activation by anionic liposomes proceeds by antibody-independent, C1q-initiated activation of the classical pathway. Purified C1q bound to anionic liposomes in an acidic lipid concentration-dependent manner. Saturation binding, but not the apparent association constant, was enhanced by increasing the cardiolipin content of the liposomes or decreasing either the pH or ionic strength of the reaction mixture. These observations indicate the involvement of electrostatic factors in the binding. A highly cationic region in the collagen-like domain of C1q comprised of residues 14-26 of the C1qA polypeptide chain was assessed for involvement in liposome binding. This region has previously been shown to mediate C1q binding to other immunoglobulin-independent activators of the classical pathway of complement. Peptides containing residues 14-26 of C1qA, denoted C1qA14-26, inhibited C1q binding to and complement activation by anionic liposomes. The inhibitory capacity of these cationic peptides had no sequence or conformation specificity. Rather, the amount of positive charge on the peptides was the determining factor. When present in excess, peptides with five cationic residues inhibited C1q binding and complement activation; however, C1q peptides with only two cationic residues did not. In addition to the C1qA14-26 region, other parts of C1q that contain cationic residues may also be involved in C1q binding to anionic liposomes.

Small-molecule complement inhibitors cannot prevent the development of the platelet storage lesion.

BACKGROUND: Suppression of the platelet (PLT) storage lesion would maintain PLT quality over longer storage times. An increased storage period would greatly improve the ability of blood agencies and hospitals to manage PLT inventories and minimize product wastage. Activation of the complement system has been proposed to play a role in initiating or potentiating the PLT storage lesion. This study examines the effect of complement inhibition on the development of the PLT storage lesion.