Activation of Fc receptors and complement by immune complexes is a common important pathogenic trigger in many autoimmune diseases and so blockade of these innate immune pathways may be an attractive target for treatment of immune complex-mediated pathomechanisms. High-dose IVIG is used to treat autoimmune and inflammatory diseases, and several studies demonstrate that the therapeutic effects of IVIG can be recapitulated with the Fc portion. Further, recent data indicate that recombinant multimerized Fc molecules exhibit potent anti-inflammatory properties. In this study, we investigated the biochemical and biological properties of an rFc hexamer (termed Fc-μTP-L309C) generated by fusion of the IgM μ-tailpiece to the C terminus of human IgG1 Fc. Fc-μTP-L309C bound FcγRs with high avidity and inhibited FcγR-mediated effector functions (Ab-dependent cell-mediated cytotoxicity, phagocytosis, respiratory burst) in vitro. In addition, Fc-μTP-L309C prevented full activation of the classical complement pathway by blocking C2 cleavage, avoiding generation of inflammatory downstream products (C5a or sC5b-9). In vivo, Fc-μTP-L309C suppressed inflammatory arthritis in mice when given therapeutically at approximately a 10-fold lower dose than IVIG, which was associated with reduced inflammatory cytokine production and complement activation. Likewise, administration of Fc-μTP-L309C restored platelet counts in a mouse model of immune thrombocytopenia. Our data demonstrate a potent anti-inflammatory effect of Fc-μTP-L309C in vitro and in vivo, likely mediated by blockade of FcγRs and its unique inhibition of complement activation.

ABSTRACT

Arabinogalactan-proteins (AGPs) are proteoglycans containing a high proportion ofcarbohydrate (typically >90%) linked to a protein backbone rich in hydroxyproline (Hyp), Ala Approximately 93% of the Pro residues are hydroxylated and hence are potential sites for glycosylation.Arabinogalactan-proteins (AGPs) occur predominantly in the intercellular spaces of plant tissues but are also associated with membranes, some cytoplasmic organelles, and the cell wall (for reviews see refs. 1-5). AGPs bind to and are precipitated by the P-glucosyl Yariv reagent (6). The function of AGPs is not established, but they may be involved in development, cell-cell interactions, and plant defense.The carbohydrate component of AGPs is generally composed of arabinose and galactose with minor amounts of other sugars. Linkage analysis is consistent with a structure based on a 3-linked (3-galactosyl backbone, branched through C(0)6 to 6-linked galactosyl side chains. The arabinose is most often present as terminal residues. The protein is usually a minor component with characteristically high levels of hydroxyproline (Hyp), Ala, and Ser (for exceptions see refs. 7 and 8). Relatively little is known about the structure ofthe protein core ofAGPs; only a few peptide sequences are available (7,(9)(10)(11) AGPs were deglycosylated using anhydrous HF (18) and fractionated by size-exclusion FPLC and RP-HPLC according to Fig. 1 D-E. The protein backbones were digested with thermolysin, and the products were separated on a C18 microbore HPLC column (Ultrasphere ODS, 2.1 x 250 mm) and eluted with a gradient of acetonitrile in 0.1% aqueous TFA. Individual peaks were repurified and sequenced (19). Amino acid analyses were performed as described by Simpson et al. (20).Isolation of cDNA Clones. A 68-base oligonucleotide, 5'-GCAAAATCACCAACAGCAACACCACCAACAGCAA-CACCACCATCAGCAGTATATAGTGAGTCGTATTA-3', was synthesized. The first part of the sequence codes for the AGP peptide A-K-S-O-T-A-T-O-O-T-A-T-O-O-S-A-V (

SummaryThis paper reports the isolation of cDNAs encoding the protein backbone of two arabinogalactan-proteins (AGPs), one from pear cell suspension cultures (AGPP¢2) and the other from suspension cultures of Nicotiana alata (AGPNa2). The proteins encoded by these cDNAs are quite different from the 'classical' AGP backbones described previously for AGPs isolated from pear suspension cultures and extracts of N. alata styles. The cDNA for AGPP¢2 encodes a 294 amino acid protein, of which a relatively short stretch (35 amino acids) is Hyp/Pro rich; this stretch is flanked by sequences which are dominated by Asn residues. Asn residues are not a feature of the 'classical' AGP backbones in which Hyp/Pro, Ser, Ala and Thr account for most of the amino acids. The cDNA for AGPNa2 encodes a 437 amino acid protein, which contains two distinct domains: one rich in Hyp/Pro, Sar, Ala, Thr and the other rich in Asn, Tyr and Sar. The composition and sequence of the Pro-rich domain resembles that of the 'classical' AGP backbone. The Asn-rich domains of the two cDNAs described have no sequence similarity; in both cases they are predicted to be processed to give a mature backbone with a composition similar to that of the "classical' AGPs. The study shows that different AGPs can differ in the amino acid sequence in the protein backbone, as well as the composition and sequence of the arabinogalactan side-chains. It also shows that differential expression of genes encoding AGP protein backbones, as well

Lectins were able to bind underlying carbohydrate structures (sialylated Tn and Forssman antigens) that are normally cryptic antigens on H-transferase transgenic mouse spleen and cardiac endothelial cells, probably as a consequence of the reduction in the electronegativity of the cell surface due to reduced sialylation. As humans have preformed anti-Tn and anti-Forssman antibodies, it is possible that these structures may become targets of the xenograft rejection process, including hyperacute rejection.

There is now considerable evidence implicating anti‐Gal xenoantibodies as a key instigator in the hyperacute rejection of discordant xenografts. As a consequence it is generally held that elimination or reduction of the Gal/anti‐Gal component is critical to overcoming hyperacute rejection. We have recently shown that in mice inactivation of the GalT gene by homologous recombination completely eliminates the expression of the Gal‐epitope and that hearts from these mice demonstrate prolonged survival when perfused ex vivo with human plasma. Unfortunately this strategy is currently not feasible in pigs because the technology to isolate porcine embryonic stem cells, which are critical for homologous recombination, is not yet available. This study investigates an alternative competition‐based transgenic strategy to suppress the level of the Gal epitope by expression of H‐transferase (α1,2‐fucosyltransferase) an enzyme which has the same substrate specificity (lactobiose) as α1,3‐galactosyltransferase. In vitro transfection of murine cells with H‐transferase reduced Gal‐epitope expression by 80–90%. A similar reduction in Gal expression was observed on PBL and thymocytes from H‐transferase transgenic mice. This reduction in Gal epitope expression resulted in a marked reduction in the reactivity of these cells with human serum. In tissues from these mice the reduction in Gal expression was inversely proportional to the endogenous level of Gal. The results of this study support pursuing this strategy as a means to reduce the xenoantigenicity of porcine tissues.

We used an ex vivo model to demonstrate that eliminating alphaGal expression further prolongs the function of mouse hearts expressing high levels of CD55 and CD59. In addition, we showed that reducing alphaGal by expressing HTF is equally as effective in prolonging CD55/CD59 heart function as knocking out Gal transferase, thus providing a feasible strategy for translating these advances to the pig.

cDNAs and corresponding genomic clones encoding a putative proline-rich protein (NaPRP3) were isolated from libraries prepared from Nicotiana alata style mRNA and genomic DNA. The predicted NaPRP3 protein is structurally similar to extensin in containing six copies of the characteristic extensin sequence Ser-Pro4, but differs in being smaller (151 residues compared with greater than 300 residues) and lacking Tyr residues. In contrast to most extensin genes, the NaPRP3 gene is not induced by mechanical wounding, and its expression is restricted to cells of the transmitting tract of the style.