It has been recognized since 1895 (1) that some gram-negative bacteria are sensitive to the lytic action of fresh serum, whereas others are highly serum resistant. In general, serum-resistant organisms are more pathogenic than serum-sensitive bacteria in animal models of infection, and serum-resistant organisms are more commonly isolated from the bloodstream of patients with gram-negative bacteremia (2). In attempts to define the basis of this important virulence factor, characteristics of the outer membrane of serum-sensitive and serum-resistant organisms have been analyzed and compared (3)(4)(5). The presence of a complete lipopolysaccharide (LPS) 1 (i.e., the smooth phenotype) is the characteristic most clearly associated with serum resistance. Rough bacteria lacking a complete LPS are almost invariably serum sensitive.The antibody and complement requirements for serum killing of bacteria also have been examined. It has been shown (6-8) that killing of gram-negative bacteria by serum requires the participation of terminal components of the complement system ((25-9). However, the mechanism of resistance of gram-negative bacteria to serum killing in the presence of adequate antibody is still unknown.Resistance to serum killing could involve the inability to form a membrane attack complex on the organism. An alternative hypothesis, however, is that a membrane attack complex that forms on the bacterial surface may be functionally impotent either because of failure to insert into the bacterial outer membrane or because the inserted complex does not cause damage to vital outer or inner membrane structures.Previous studies have examined a number of aspects of this issue. Studies have suggested that serum-sensitive and serum-resistant strains of Escherichia coli (9, 10) or Salmonella typhimurium (11) have equivalent amounts of C3 deposited. It is not resolved whether (25 is deposited on serum-resistant bacteria. Reynolds et al. (11) could not demonstrate deposition of functional C5 on serum-resistant S. typhimurium in Mg ++ saline after incubation in C6-deficient rabbit serum. On the other hand, Ogata and Levine (10) demonstrated equivalent (25 consumptior~ by strains of E. coli that varied in complement sensitivity; however, evidence for levels of cell-bound (25 was not 1 Abbreviations used in this paper: CFU, colony-forming unit; HBSS, Hanks' balanced salt solution; LPS, lipopolysaccharide; PNHS, pooled normal human serum; RT, room temperature. J. Exp. MED.

The mechanism for consumption of terminal complement components and release of bound components from the surface of serum-resistant salmonella minnesota S218 was studied. Consumption of C8 and C9 by S218 occurred through interaction with C5b67 on the bacterial surface because C8 and C9 were consumed when added to S218 organisms previously incubated in C8-deficient serum and washed to remove all C5b67 on the bacterial surface because C8 and C9 were consumed when added to S218 organisms previously incubated in C8- deficient serum and washed to remove al but cell bound C5b67. Rapid release of (125)I C5 and (125)I C7 from the membrane of S218 was dependent on binding of C8 because (125)I C5 and (125)I C7 deposition in C8D serum was stable and was twofold higher in C8D than in PNHA, and addition of purified C8 or C8 and C9 to S218 previously incubated in C8D serum caused rapid release of (125)I C5 and (125)I C7 from the organism. Analysis by sucrose density gradient ultracentrifugation of the fluid phase from the reaction of S218 and 10 percent PNHS revealed a peak consistent with SC5b-9, in which the C9:C7 ratio was 3.3:1, but the NaDOC extracted bound C5b-9 complex sedimented as a broad peak with C9:C7 of less than 1.2:1. Progressive elution of C5b67 and C5b-9 from S218 but not serum-sensitive S. minnesota Re595 was observed with incubation in buffers of increasing ionic strength. Greater than 90 percent of the bound counts of (125)I C5 or (125)I C9 were released from S218 by incubation in 0.1 percent trypsin, but only 57 percent of (125)I C9 were released by treatment of Re595 with trypsin. These results are consistent with the concept that C5b-9 forms on the surface of the serum-sensitive S. minnesota S218 in normal human serum, but the formed complex is released and is not bactericidal for S218 because it fails to insert into hydrophobic outer membrane domains.

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A method is described for the separation of blood and bone marrow leukocytes on the basis of buoyant density, using a discontinuous Ficoll-Hypaque density gradient. The median cell densities of monocytes and lymphocytes were found to be 1.067–1.077 and 1.073-1.077 g/ml, respectively. The cells of the myeloid series were shown to increase in density with maturation; the myeloblasts had the lowest density (1.064- 1.065 g/ml) and the neutrophils the highest (greater than 1.080 g/ml). Cell volumes have been determined on isolated cell populations. The findings were: monocytes, 534 +/- 47 cu mu; lymphocytes, 247 +/- 18 cu mu; and neutrophils, 468 +/- 24 cu mu. Fourteen patients with acute lymphoblastic leukemia were studied. In four patients, the lymphoblasts were of low density (less than 1.068 g/ml), whereas the remaining patients had high density (greater than 1.068 g/ml) lymphoblasts. These four patients had large numbers of lymphoblasts in the peripheral blood and a poor prognosis. Lymphoblast volumes were not different in these two groups and were unrelated to prognosis.

The production of antibodies after immunization requires antigen recognition by B cells, expansion of the pool of antigen-reactive B cells, and finally, differentiation of these B cells into antibody-secreting plasma cells. Current models for this process of B cell maturation in humans involve separate signals for B cell activation, proliferation, and differentiation (1, 2). One ofthe lymphokines that has been shown to enhance B cell proliferation is a 50-60-kD product of activated T cells and of the Namalwa cell line termed high molecular weight B cell growth factor (HMW BCGF)t (3). Human HMW BCGF has been purified to homogeneity, and this molecule has no obvious analog among the B cell growth factors of mice .The environment of an ongoing immune response often includes the activated products of various inflammatory cascades, such as clotting or complement proteins .Multiple complement activation fragments have been shown to modulate the immune response, including C3a, C5a, C3b, C3d, and Ba (4-9) . Engagement of B cell receptors for C3b (CRI) and C3d (CR2) by antibodies can also influence B cell proliferation (10, 11) . The possibility that Bb can affect immune responses in vitro has been suggested (12) but has not been systematically investigated . In the present work, we have studied the modulatory influence of the complement activation frag-