The interaction between human fibronectin and 17 strains of staphylococci was studied in an attempt to elucidate the staphylococcal cell wall component(s) involved in fibronectin binding and to determine the influence of fibronectin upon phagocytosis by three types of phagocytic cells. Purified, radiolabeled fibronectin bound to a similar degree to six laboratory strains and three fresh clinical isolates of Staphylococcus aureus; similar binding of fibronectin was found with S. aureus strains deficient in cell wall teichoic acid or clumping factor and coagulase, as well as with three strains of S. epidermidis. There was minimal binding of fibronectin to encapsulated S. aureus and to Escherichia coli. Fibronectin bound to intact cells and to a crude cell wall preparation of S. aureus H, but not to purified cell walls or peptidoglycan. Trypsinization of staphylococci prevented subsequent fibronectin binding, but binding did not correlate well with the protein A content in S. aureus cell walls. At physiological concentrations, fibronectin binding to staphylococci did not promote phagocytosis of bacteria by human polymorphonuclear leukocytes, monocytes, or alveolar macrophages. Also, depletion of fibronectin from normal human serum did not result in a measurable loss of opsonic activity for staphylococci. It is concluded that fibronectin binding to staphylococci involves a surface protein shared among strains of S. aureus and S. epidermidis, and that in comparison to C3b and IgG, fibronectin plays a relatively minor role as an opsonin for staphylococci.

A B S T R A C T Human alveolar macrophages (AM

Phagocytosis and killing by alveolar macrophages from humans, rabbits, rats, and hamsters were compared in vitro. In the absence of serum opsonins, human alveolar macrophages could phagocytize Staphylococcus aureus Cowan I (protein A positive), but not S. aureus EMS (protein A negative) or Pseudomonas aeruginosa MN. In contrast, rabbit, rat, and hamster alveolar macrophages did not phagocytize S. aureus Cowan I or other nonopsonized bacteria. Human alveolar macrophages, but not other species, stained positively with fluorescein isothiocyanate-conjugated protein A. When opsonized bacteria were studied, phagocytosis by human, rabbit, and hamster alveolar macrophages was found to be mediated by both Fc and C3 receptors. However, only Fc receptor-mediated phagocytosis of bacteria was demonstrated for rat alveolar macrophages. Differences were also found in the kinetics of bacterial killing by alveolar macrophages from different species. Human and rabbit alveolar macrophages rapidly killed opsonized S. aureus Cowan I. However, bacterial killing by hamster alveolar macrophages proceeded at a slower rate, and rat alveolar macrophages completely failed to kill S. aureus. These significant differences in the function of alveolar macrophages from four different species emphasize the need to document the appropriateness of animal models before using them to predict the biological activities of human alveolar macrophages.

Previous studies of encapsulated Staphylococcus aureus have shown that the opsonins of normal, nonimmune human serum (complement factor C3 and IgG) bind beneath the capsule, i.e., on the cell wall, and when bound at this site these opsonins are not effective in promoting phagocytosis of the bacteria by polymorphonuclear leukocytes (PMN). In this investigation immune antibody was added to human serum to effect opsonization of encapsulated S. aureus. Opsonization was assessed by quantitating the uptake of 3H-labeled staphylococci by human PMN, and the amount of C3 fixation to bacteria was measured in a quantitative fluorescent immunoassay. Low levels of immune antibody (IgG) effectively opsonized encapsulated S. aureus when added to fresh but not to heated serum; phagocytosis of the staphylococci was mediated via pronase-sensitive membrane receptors (presumably C3b receptors) of PMN. Experiments with C2-, C3-, or C5-deficient human sera revealed that C3 was required for opsonization and that activation of C3 was mediated via the alternative complement pathway. Encapsulated S. aureus bound significantly less C3 than unencapsulated strains in diluted normal serum; addition of immune antibody, however, increased C3 fixation 4.7-fold (p less than 0.005). Immunoelectron microscopy localized C3 throughout the capsule as well as on the staphylococcal cell wall when bacteria had been opsonized in human serum with immune antibody. Without immune antibody, C3 binding was restricted to the cell wall. At approximately 10-fold higher levels of immune antibody, opsonization and phagocytosis of encapsulated S. aureus was independent of complement and pronase-sensitive receptors on PMN. These studies show that, in addition to immune antibody, the alternative pathway of complement plays an important role in the opsonization of encapsulated S. aureus strains and suggest that complement may be crucial to the in vivo clearance of these organisms.

IntroductionHyperacute (HAR) and delayed xenograft (DXR) rejection are well defined barriers following discordant transplantation of porcine organs into primate recipients. Significant progress has been made in recent years to overcome these immunological barriers, based on the development of pigs genetically modified to reduce immunogenicity: [1,2] A major step has been the generation of pigs lacking expression of Gala1,3Gal (GalTKO), the principal carbohydrate target of human anti‐pig antibody. [3,4] However, both in vitro and in vivo studies demonstrated that non‐gal antibodies have the ability to activate endothelial cells, and thus, amplify procoagulant molecular incompatibilities between pig and primate.[5,6] Based on our prior work with pigs expressing the Gal epitope, we tested the hypothesis that over‐expression of human complement regulatory proteins (hCRP) and/or blockade of the CD154/CD40 costimulation pathway will result in reduced graft injury, anti‐pig responses and coagulation dysregulation. Briefly, twenty two organs (13 hearts, 9 kidneys) from GalT‐KO pigs (9 expressing hCRP) were transplanted into baboons that received no immunosuppressive therapy, or a therapeutic regimen based on costimulation blockade. The incidence of HAR was decreased in GalTKO organs expressing hCRP as compared to GalTKO. Pre‐transplant anti‐non‐Gal antibody titers were inconsistently associated with early GalT‐KO xenograft failure. In contrast, coagulation cascade activation and platelet activation correlated closely with incidence of HAR of GalTKO organs. Strong elicited anti‐non‐gal antibody responses were detected after transplantation of a GalTKO organ. Delayed xenograft rejection was associated with coagulation dysregulation and elicited anti‐non‐gal antibody production. Future work in xenotransplantation should place emphasis on further targeting of anti‐non gal humoral immune responses and inhibiting coagulation activation. References 1. Pierson RN III. JAMA 2009; Current status of xenotransplantation. 30: 967–969. 2. Cooper DK, Dorling A, Pierson RN III, et al. Alpha1,3‐galactosyltransferase gene‐knockout pigs for xenotransplantation: where do we go from here? Transplantation 2007; 84: 1–7. 3. Yamada K, Yazawa K, Shimizu A, et al. Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3‐galactosyltransferase gene‐knockout donors and the cotransplantation of vascularized thymic tissue. Nat Med 2005; 11: 32–34. 4. Kuwaki K, Tseng YL, Dor FJ, et al. Heart transplantation in baboons using alpha1,3‐galactosyltransferase gene‐knockout pigs as donors: initial experience. Nat Med 2005; 11: 29–31. 5 Saethre M, Baumann BC, Fung M, Seebach JD, Mollnes TE. Characterization of natural human anti‐non‐gal antibodies and their effect on activation of porcine gal‐deficient endothelial cells. Transplantation. 2007; 84: 244–250. 6. Hisashi Y, Yamada K, Kuwaki K, et al. Rejection of cardiac xenografts transplanted from alpha1,3‐galactosyltransferase gene‐knockout (GalT‐KO) pigs to baboons. Am J ...