Multimeric C9 within C5b-9 is required for inner membrane damage to Escherichia coli J5 during complement killing

We have shown recently that an average of three or more C9 molecules must bind to C5b-8 on Escherichia coli strain J5 to cause direct complement killing in the absence of serum lysozyme. We initially confirmed and extended this observation by showing that deposition of a large number of C5b-9 complexes bearing 1C9 per C5b-8 was not bactericidal for J5. To identify the target site for bactericidal C5b-9 deposition, we measured release of periplasmic and cytoplasmic markers of different size from J5 as the C9:C5b-8 ratio was changed, because the diameter of the C5b-9 channel is known to increase as the C9:C5b-8 ratio increases. To facilitate measurement of release of the periplasmic marker beta-lactamase (BLA), J5 was transformed for high level constitutive TEM-1 BLA production (J5-Amp). Multimeric C9 within C5b-9 (C9:C5b-8 greater than 3) was required to release BLA (m.w. 28,900) from J5-Amp regardless of whether cells bore 310, 560, or 890 C5b-9/organism. Curves of both BLA release and killing vs C9:C5b-8 ratio were sigmoidal and nearly superimposable. Release of the small cytoplasmic marker 86Rb, a potassium analog, also required a minimum C9:C5b-8 ratio of 3:1; specific 86Rb release did not occur in the absence of killing. Release of the large cytoplasmic marker beta-galactosidase (m.w. 505,000) did not occur even at the highest achievable C9:C5b-8 ratio of 11:1, despite greater than 99.9% killing, indicating that there was no dissolution of the peptidoglycan layer due to incomplete removal of serum lysozyme. Complement-mediated killing of J5 requires sufficient damage to the outer membrane or formation of a sufficiently large C5b-9 channel to release the large periplasmic marker BLA. The requirement of multimeric C9 for 86Rb release suggests that at low C9:C5b-8 ratios, either C5b-9 does not have access to the cytoplasmic space or that the J5 K+ transport systems are able to compensate for putative C5b-9 channels.     

Activated terminal complement in cerebrospinal fluid in Guillain-Barré syndrome and multiple sclerosis

A quantitative enzyme-linked immunosorbent assay was used to measure the concentration of fluid-phase complement C5b-9 complexes (SC5b-9) in the cerebrospinal fluid (CSF) of 14 patients with acute monophasic Guillain-Barré Syndrome (GBS), 21 patients with multiple sclerosis (MS), and 11 patients with noninflammatory central nervous system (CNS) diseases. SC5b-9 complexes were detected in the CSF of 13 of 14 patients with acute GBS (mean, 3.08 micrograms/ml; range, 0 to 7.1 micrograms/ml) and 16 of 21 patients with MS (mean, 1.83 micrograms/ml; range, 0 to 7.5 micrograms/ml). In the control group of patients with noninflammatory CNS diseases, SC5b-9 was not detected in eight of 11 and was present in low concentrations in the remaining three patients (mean, 0.28 micrograms/ml; range, 0 to 1.7 micrograms/ml). The finding of SC5b-9 complexes in the CSF of patients with GBS and MS suggests that terminal complement components may participate in the tissue-damaging processes in these diseases.     

Analysis of chromosome aberrations in lymphocytes of long-term heavy smokers

We assessed the incidence of structural chromosome aberrations in 500 diploid first-division metaphases from 48-h lymphocyte cultures from each of 6 non-smokers and from 6 persons who had smoked a minimum of 1 pack of cigarettes per day for at least 20 years. Cytogenetic analyses of coded slides revealed a single dicentric chromosome with its accompanying fragment and two symmetrical chromatid exchanges in 3000 metaphases from the non-smokers. In contrast, 9 dicentric chromosomes, 8 translocations or inversions, and 7 chromatid exchanges were observed in 3000 metaphases from lymphocyte cultures from the 6 heavy smokers. A total of 13 metaphases having chromosome-type inter- or intra-changes was noted including 9 with a single aberration, and 4 with 2 or more. Our findings provide additional evidence of the in vivo clastogenicity of cigarette smoke in long-term heavy smokers, and further demonstrate that the distribution of chromosome-type exchange aberrations is overdispersed relative to that expected based on Poisson assumptions.     

The biologic activity of mast cell granules. IV. The effect of complement depletion on rat cutaneous late phase reactions

Cutaneous late phase reactions (LPR) in rats can be induced by the intradermal injection of anti-IgE antibody or isolated rat peritoneal mast cell granules. Rat LPR are characterized by neutrophil-rich infiltrates at 2 to 8 hr followed by mononuclear cell-rich infiltrates thereafter. Rat Arthus reactions are histologically similar and are complement (C) dependent. To determine the importance of C in the pathogenesis of rat LPR compared with its role in Arthus reactions, rats were treated with cobra venom factor (CVF) (250 U/kg i.v.), and the effects of this treatment on total hemolytic complement (CH50), C3 titers, LPR, and Arthus reactions were assessed. CVF treatment produced profound decreases in both CH50 (from 197 +/- 20 to less than 1.0 U/ml) and C3 (from 44,240 +/- 2840 to less than 5 U/ml) titers after 6 hr, which persisted through at least 30 hr. The inflammatory intensity of heterologous reverse passive Arthus reactions was significantly decreased in CVF-treated animals. In contrast, the intensity of LPR was unaffected by CVF treatment. Therefore, although LPR and Arthus reactions share certain histologic characteristics, these similarities are not due to a mutual requirement for the presence of C.     

Studies on the mechanism of bacterial resistance to complement-mediated killing. VI. IgG increases the bactericidal efficiency of C5b-9 for E. coli 0111B4 by acting at a step before C5 cleavage

Our previous experiments showed that immune IgG and F(ab')2, but not Fab', mediated serum killing of Escherichia coli 0111B4, strain 12015 (12015), without significantly increasing the extent of terminal complement (C) component attachment to the bacterial surface. We concluded that bactericidal antibody must change either the site or the nature of C5b-9 bacterial attachment. To pursue this possibility, conditions necessary for elution of C5b-9 from the bacterial surface were examined. Forty-two to 44% of 125I-C9 was released from the serum-resistant nonpresensitized 12015 by 1 M NaCl or 0.1% trypsin, compared with the 21 to 24% release from the serum-sensitive presensitized isolate under the same condition. When strain 12015 bearing 125I-C9 was lysed in a French pressure cell, 73.1% of 125I-C9 was released with the capsular fraction if the organisms had not been presensitized. In contrast, on presensitized 12015, 70.2% of 125I-C9 remained associated with the outer membrane after such lysis. These results suggested that C5b-9 was trapped within or underneath the capsule of 12015 in the absence of bactericidal antibody, but that addition of antibody led to C5b-9 insertion into the outer membrane with bacterial killing. The requirement of C components preceding C5 for bacterial killing was next examined. Minimal killing of presensitized 12015 occurred when a terminal C complex was formed by acid activation from purified C5, C6, C7, C8, and C9 in the absence of C3 or earlier components. In contrast, between 1.2 and 3 log killing of nonpresensitized rough Salmonella minnesota and rough E. coli was observed in the same system. Killing of 12015 was examined with bacteria incubated in C5-deficient serum (C5D), followed by washing and the addition of purified C5, C6, C7, C8, and C9 to permit C5b-9 formation. Antibody was added before or after incubation in C5D serum, or after the addition of purified C5-C9. Under conditions of equivalent C3 and C9 binding, significant killing occurred only when antibody was added before incubation in C5D serum. These results show that antibody must be present at or before the time of C5 convertase formation to mediate killing of 12015 by C5b-9. Therefore, antibody is unlikely to be functioning primarily to alter the bacterial surface to expose sites for C5b-9 insertion, nor is the effect of antibody simply to increase C3 and terminal component binding. We postulate that antibody mediates killing of 12015 by localizing C5b-9 around antibody-clustered sites of C3 and C5 convertase formation.     

Characterization of host cell-derived membrane proteins of the vacuole surrounding different intracellular forms of Trypanosoma cruzi in J774 cells. Evidence for phagocyte receptor sorting during the early stages of parasite entry.

Trypanosoma cruzi, an obligate intracellular protozoan parasite, exhibits developmental regulation of virulence. Although both noninfective epimastigote and infective trypomastigote stages of T. cruzi enter phagocytic cells via the formation of a parasitophorous vacuole (PV), only the latter developmental stages survive ingestion and perpetuate the infection. To determine whether the membrane composition of PV surrounding these different stages might contribute to differences in the outcome of infection, we identified selected membrane constituents by immunofluorescence and intracellular radioiodination, and studied their incorporation into PV. Complement receptors (CR3) are incorporated preferentially into the PV membrane surrounding serum-opsonized epimastigotes but not culture-derived metacyclic trypomastigotes. FcR are not preferentially incorporated into PV membranes unless epimastigotes or culture-derived metacyclic trypomastigotes are opsonized with anti-T. cruzi antibody. PV surrounding either parasite stage contain beta 1 integrins and lysosomal membrane glycoproteins (lgp). These results indicate that the plasma membrane glycoproteins incorporated into the surrounding PV membrane differ depending upon the stage of parasite being internalized, and that these differences reflect, at least in part, selective ligation of cell surface receptors mediating uptake. Furthermore, they imply that although virulent trypomastigote stages may avoid host cell uptake by conventional phagocytic receptors, i.e., CR3 or FcR, they do not escape fusion with an lgp-containing vacuole where they could still be exposed to lysosomal antimicrobial mechanisms.     

Neutron RBEs for mouse skin at low doses per fraction

The effect of low doses of 240 kVp X rays or of 3 MeV neutrons has been investigated using skin reactions on mouse feet as the biological system. Eight or nine repeated small doses of radiation were used, followed by graded "top-up" doses to bring the reactions into a detectable range. By comparing dose-response curves, the RBE has been determined for neutron doses per fraction ranging from 0.25-1.0 Gy. The data are consistent with a limiting RBE of between 7 and 10 at very low doses. A review of other published RBE values for low doses per fraction shows a wide range of RBEs . Very few studies show a plateau value for the RBE. These findings are more consistent with dose-response data that fit a linear-quadratic model than with a multitarget single-hit model.     

Evaluations of cellular proliferation and chromosome breakages after in vitro exposure of human lymphocytes to calcium or zinc DTPA

The analysis of mitotic indices (MI) and chromosome breakages in metaphases of 50-hr lymphocyte cultures exposed to the calcium or zinc chelates of diethylenetriamine pentaacetic acid (DTPA) demonstrated: (1) an 80% reduction in MI in cultures from three women but no reduction in those from two men after in vitro exposure to CaDTPA in concentrations as low as 10 micrograms/ml culture medium, and complete suppression of mitoses in cultures from men and women after exposure to 40 micrograms/ml CaDTPA; (2) minor suppression in MI in cultures from women and none in those from men after exposure to 40 or 80 micrograms/ml ZnDTPA; (3) no ring or dicentric chromosomes in 1700 metaphases from DTPA-treated cultures. Likewise, in other experiments we observed no differences in the frequency or distributions of rings and dicentrics in lymphocyte cultures from two persons after in vitro exposure to 250-R 60Co gamma radiation in the presence or absence of 10 micrograms/ml CaDTPA or 10 or 80 micrograms/ml ZnDTPA. These data indicate that while accurate estimates of the frequencies of radiation-induced rings and dicentrics in lymphocytes can be made in actinide-contaminated persons undergoing DTPA chelation therapy, blood samples for cytogenetic cultures should not be obtained from chelated patients until the compound has been cleared from the blood plasma.