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The glomerular basement membrane was subjected to digestion with specific enzymes to determine the chemical nature (sialoglycoproteins, collagenous peptides, or glycosaminoglycans) of the anionic sites previously demonstrated in the laminae rarae. Enzyme digestion was carried out both in situ and in vitro. Kidneys were perfused in situ with enzyme solutions followed by perfusion with fixative containing the cationic dye, ruthenium red, to detect the anionic sites. Glomerular basement membranes were isolated by detergent treatment of glomeruli and incubated with enzyme solutions, followed by incubation with cationized ferritin (pI 7.3-7.5) to label the anionic sites. Only highly purified enzymes free of proteolytic activity were used. Thefindings were the same both in situ and in vitro. The anionic sites were unaffected by treatment with neuraminidase, chondroitinase ABC, and tes-ticular or leech hyaluronidase. However, they could no longer be demonstrated after digestion with crude heparinase, purified heparitinase, or Pronase or after nitrous acid oxidation. The results demonstrate that the sites contain heparan sulfate since they are removed by treatment with heparitinase and by nitrous acid oxidation-procedures specific or heparan sulfate; and that sialoglycoproteins or other glycosaminoglycans do not represent ma'or components of these sites since the latter are not affected by digestion with neuraminidase and other gly cosaminoglycan-specific enzymes. Identical findings were o, tained on basement membranes in other locations (Bowman's capsule, tubule epithelium and endothelium of peritubular capillaries). The presence of heparan sulfate in the glomerular basement membrane is discussed in relation to the charge-selective properties of the glomerular filter and in relation to its potential involvement in various types of glomerular injury. Physiologic studies have established that the glomerular cap-illaries function as a size-and charge-selective barrier in the production of the glomerular filtrate and in the retention of plasma proteins in the circulation (1-3). By inference it has been assumed (1-3) that charge selectivity is due to the presence in the glomerulus of fixed negative charges, but their precise nature and location in the capillary wall have not been established. Since macromolecular compounds carrying sialic acid residues have been-up to recently-the only known polyanions present in the glomerulus, they have been considered the most likely candidates responsible for the charge barrier (1, 2, 4-7). Sialyl residues have been found in all three layers of the capillary wall. They have been detected in isolated glomerular basement membranes (GBM) (8-10) and in association with the cell membranes of both the endothelium (11-12) and epithelium (4-7, 11), where they are part of the sialic acid-rich, cell-surface coats. A colloidal iron-stainable, cell-coat material is particularly concentrated on the epithelium. Accordingly, it has been repeatedly suggested (1, 2, 4-7) that this "epithelial polyanion"...

Encapsulated Staphylococcus aureus strains are more virulent than unencapsulated staphylococci, and this phenomenon has been associated with decreased opsonization of encapsulated bacteria by normal human serum. Peptidoglycan, a major cell wall component of S. aureus, has been shown to promote opsonization of this bacterial species by certain components of the serum complement system. However, when the processes of complement activation and opsonization of encapsulated staphylococci have been studied, it has been found that encapsulated bacteria activate complement efficiently and C3 is bacteria associated, yet these organisms are not efficiently phagocytized by human polymorphonuclear leukocytes. In this study, the hypothesis was tested that opsonically active molecules are not on the true external surface of encapsulated organisms but rather are cell wall associated and thus "hidden" from human polymorphonuclear leukocytes. By using immunoelectronmicroscopy, C3 was found to be localized on the cell wall of an encapsulated S. aureus strain after incubation of the organism in normal human serum. When shrinkage of the capsule was prevented by treatment with anticapsular antibody, the C3 was again shown to be cell wall associated and located beneath the bacterial capsule. These results suggest that encapsulated S. aureus may resist phagocytosis because opsonically active C3 molecules are not exposed at the true external surface of the bacterium.

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This study evaluated a variety of fixatives and methods of tissue preparation for application cf the direct Tube Numbers Tubenumber-a'25 30 35 40 Stain intensity-a. 442 SISSON, VERNIER of fixation on tissue antigens. Alternate blocks from the same kidneys were fixed by immersion for variable time periods. The fixatives evaluated included: phosphate-buffered formalin (4); glutaraldehyde (1, 2, and 0.5' i) in 0.05 M sodium cacodylate (SC) buffer (SCB); paraformaldehyde 4Cfl with glutaraldehyde (5#{176}7i)as described by Karnovsky (23), and a 1:4 dilution of this fixative in SC; paraformaldehyde with picric acid (46), paraformaldehyde (F/c) in sodium phosphate buffer (4 1 1, and a modification of the paraformaldehyde

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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.