Complement activation during exposure of plasma to cuprophan has been postulated to cause leukopenia and hypoxia in hemodialysis patients. To determine if hypoxia is related to leukopenia and if complement activation leads to a depletion of functional complement components, we dialyzed four patients three times sequentially against each of four types of membranes: cuprophan, regenerated cellulose, cellulose acetate, and polyacrilonitrile. Within 20 min there was a marked leukopenia with cuprophan from 5541 +/- 376 to 1216 +/- 94 (P less than 0.001) and with regenerated cellulose from 5541 +/- 411 to 1533 +/- 203 (P less than 0.001). With cellulose acetate, the change from 5558 +/- 400 to 3783 +/- 341 (P less than 0.001) was less dramatic, and with polyacrilonitrile the fall from 5591 +/- 381 to 464 +/- 401 (P less than 0.02) was minimal. After 2 and 4 hours of dialysis, a rebound leukocytosis was seen with cuprophan, regenerated cellulose, and cellulose acetate, but not with polyacrilonitrile. Transient thrombocytopenia occurred with cuprophan and regenerated cellulose. In spite of the variable degree of leukopenia, all membranes induced a similar and significant hypoxia, which was progressive throughout dialysis, even during the rebound leukocytosis. After 4 hours, the mean PO2 ranged from 91 to 93 mm Hg with all membranes. Functional hemolytic titers of whole complement, C3, C5, and C4 were normal prior to hemodialysis and failed to decrease after 4 hours with any membrane. It is concluded that hemodialysis leukopenia is membrane-dependent and is not the cause of hypoxia. In addition, hemodialysis complement activation does not lead to functional complement depletion and is of no clinical significance.
Serum bacteriostasis of Staphylococcus aureus was characterized quantitatively and qualitatively. Bacteriostasis was proportional to the concentration of serum. Reproducibility was good; freezing and thawing did not materially affect the end point. Four of six different strains, including the propagating S. aureus strain for phage 73 which does not produce coagulase, were susceptible to serum bacteriostasis in similar titers; two were not susceptible at all. All six strains were effective inhibitors of bacteriostasis. Active and inactive coagulase were also inhibitors. In contrast to sensitive S. aureus, S. epidermidis and Streptococcus salivarius were not uniformly susceptible to bacteriostasis by different serums. Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Salmonella montevideo, S. zymogenes, and Diplococcus pneumoniae were not susceptible. Among gram-positive bacteria, only D. pneumoniae inhibited S. aureus bacteriostasis. Agglutinins of S. aureus and nonspecific substances such as lysozyme, 3-lysin, C-reactive protein, and transferrin were not responsible for S. aureus serum bacteriostasis. After diethylaminoethyl column fractionation of serum, the bacteriostatic principle was eluted in proximity to blood group antibody; immunoglobulins A, G, and M appeared to be present in bacteriostatic fractions. It is suggested that S. aureus bacteriostasis by serum is due to natural antibody and that inhibitory reactions with pneumococci and coagulase are due to common antigens.
Functionally pure C8 from human or guinea pig serum was inactivated by 1 to 2 × 10-3 M EDTA. Native C8 in serum was partially inactivated by EDTA, but much longer periods of incubation at 37°C were required for this to be evident. The effect of EDTA on C8 was dependent upon time, temperature, and concentrations of both the chelating agent and C8. The inactivation of C8 by EDTA was partially or totally reversed by the addition of Ca2+ or by dialysis, but reversal was dependent upon the duration of exposure and the temperature. The process became irreversible within relatively short periods of time at 30°C and 37°C. EDTA had no apparent effect on the activity of cell-bound C8 or on the binding site of C8 for C9, but it did impair the binding site of C8 for C7. Neither EAC1–7 nor C9 was affected by EDTA.
When applied to human serum, the methods previously described by Nelson, Rommel and Stolfi for the preparation of a mixture of complement (C′) components from guinea pig serum suitable for the titration of C′8 and C′9 proved unsatisfactory. The reagents so obtained were contaminated with considerable quantities of C′8 and C′9 and were deficient in C′2. Three methods have been developed to separate a suitable human complement reagent mixture, using serum essentially free of C′1 as starting material. 1) Column chromatography on carboxymethyl (CM)-cellulose at 0.07 M relative salt concentration (RSC), pH 5.0, followed by diethylaminoethyl (DEAE)-cellulose at 0.16 M RSC, pH 7.5; 2) hydroxylapatite chromatography at 0.10 M PO4, pH 6.9, followed by Sephadex G-200 gel filtration; 3) DEAE-cellulose chromatography at 0.035 M RSC, pH 7.0, followed by G-200 gel filtration. These methods have yielded reagents containing sufficient C′4, C′2, C′3, C′5, C′6 and C′7 for the generation of the stable cellular intermediate (EAC 1423567).
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