Tumor necrosis factor/cachectin. Induction of hemorrhagic necrosis in normal tissue requires the fifth component of complement (C5).

TNF induces hemorrhagic necrosis (HN) when injected into skin exposed to bacterial agents but not when injected into normal skin. In this paper, we present several lines of evidence suggesting that TNF requires the fifth component of complement (C5) to induce HN in skin exposed to bacteria. First, mouse strains that do not have C5 did not develop HN after injection of TNF and bacteria into skin. Second, plasma from C5-sufficient mice could correct the defect in these C5-deficient mice. Third, heating at 56 degrees C for 30 min inactivated the capacity of plasma to reconstitute C5-deficient mice. Fourth, CVF, which is known to inactivate complement, abrogated the capability of C5-sufficient mice to respond. Fifth, depleting plasma of hemolytic activity while generating C5a did not affect the capacity of the activated plasma to reconstitute C5-deficient mice. Finally, only the plasma fraction containing molecules of the size range of C5a reconstituted C5-deficient mice. These findings indicate that C5a and not the membrane attack complex is required for HN. Although we do not know through which mechanism C5a participates in the development of HN, we propose that the described HN response is related to a local defense mechanism in which TNF and C5a lead to the disruption of capillaries in the direct vicinity of bacteria. By this mechanism the rapid spread of bacteria or their products into the circulation is prevented. Such a tissue response is consistent with the known higher susceptibility of C5-deficient mice to bacterial infections and provides a model with which to search for the multiple steps involved in this important local defense mechanism.

Hormonal control of lysosomal enzyme release from human neutrophils. Effects of autonomic agents on enzyme release, phagocytosis, and cylic nucleotide levels.

The purpose of this investigation was to examine the effects of autonomic neurohormones, cyclic nucleotides, and related agents on the immunologic discharge of lysosomal enzymes from, and phagocytosis by, purified human neutrophils. In order to discern the possible intracellular mechanisms by which certain neurohormones influence neutrophil function, the concentrations of cyclic AMP and cyclic GMP in neutrophils were assessed during cell contact with phagocytizable particles and autonomic agents. The model system employed for study was the interaction of purified human neutrophils with rheumatoid arthritic (RA) serum-treated zymosan particles at 37 degrees C in a neutral, balanced salt solution containing glucose. Neutrophils ingested the particles and discharged beta-glucuronidase but not lactate dehydrogenase activity during 30 min of incubation. Treatment of zymosan particles with RA serum was more effective than treatment with normal serum with regard to the extent of both particle uptake and lysosomal enzyme release. During contact of neutrophils with RA serum-treated zymosan particles epinephrine, isoproterenol, and cyclic AMP inhibited both particle ingestion and beta-glucuronidase discharge. These actions of epinephrine were associated with a concomitant elevation of cyclic AMP levels. In contrast to the actions of catecholamines and cyclic AMP, acetylcholine and cyclic GMP accelerated lysosomal enzyme release without affecting particle uptake. The actions of acetylcholine were associated with a concomitant elevation of cyclic GMP levels. Increases in neutrophil levels of cyclic GMP but not of cyclic AMP were associated also with the discharge of beta-glucuronidase provoked by particles in the absence of added cholinergic agents. The data suggest that the immunologic release of lysosomal enzymes from human neutrophils can be regulated by autonomic neurohormones, perhaps via the selective formation of appropriate nucleotides.

L-ficolin binding and lectin pathway activation by acetylated low-density lipoprotein.

L-ficolin, like mannan-binding lectin (MBL), is a lectin pathway activator present in normal human plasma. Upon binding ligand, l-ficolin similarly initiates C4 cleavage via the serine protease MBL-associated serine protease-2 (MASP-2). We sought further insight into l-ficolin binding reactions and MASP-2 activation by passing plasma through GlcNAc-derivatized Sepharose. l-Ficolin bound in 1.0 M NaCl-ethylenediamine tetraacetic acid (EDTA), and remained bound in NaCl-free EDTA, while MASP-2 eluted in proenzyme form ( approximately 20% yield, > 40 000-fold purification). L-Ficolin was eluted with GlcNAc in 1.0 M NaCl ( approximately 10% yield, > 3000-fold purification), with trace amounts of C3, alpha(2)-macroglobulin and both native and activated MASP-2. These preparations were utilized to investigate l-ficolin reactivities with acetylated low-density lipoprotein (A-LDL) as a model ligand in albumin-free systems. L-Ficolin bound strongly to A-LDL in the absence as well as presence of calcium, including saline-EDTA, and was optimal in 1.0 M NaCl-EDTA, but binding failed to occur in EDTA in the absence of NaCl. The addition of l-ficolin to immobilized A-LDL resulted in activation of MASP-2 in unmodified but not ficolin-depleted plasma unless l-ficolin was restored. We conclude that A-LDL is a useful ligand for investigation of l-ficolin function; both binding and activation are optimally examined in systems free of albumin; and ligand binding in 1.0 M NaCl in EDTA can be useful in the isolation of l-ficolin and native MASP-2.

Inherited deficiency of the seventh component of complement associated with nephritis. Propensity to formation of C56 and related C7-consuming activity.

A 46-yr-old female with chronic pyelonephritis was found to lack complement (C) activity by the use of hemolytic screen assays in agarose gels. These assays also revealed a propensity of patient serum to form an activated complex of the fifth and sixth components of C, C56. Each of the C component hemolytic activities was present in normal or elevated amounts with the exception of C7, which was undetectable; addition of purified C7 led to the restoration of hemolytic activity. C-dependent phagocytosis, immune adherence, and neutrophil chemotaxis were normal. Family studies demonstrated that the defect was transmitted as an autosomal codominant apparently not linked with alleles at the HLA-A or HLA-B loci. Persisting C56 was readily formed in this as compared to normal serum upon incubation with multiple C activators including zymosan, inulin, immune complexes, heat-aggregated human gamma globulin, endotoxin, and agarose. A heat-stable (56 degrees C, 30 min) activity which consumed C7 with time-and temperature-dependent kinetics was detected in plasma and serum, and seemed to be similar to a "C7 inactivator" previously described in another C7-deficient individual. However, this activity was found to have properties identical to those of C56 during low ionic strength precipitation and chromatography on Sephadex G-200, to be specifically removed upon passage through an anti-C5 immunoadsorbent column, and to be associated with a small amount of C56, suggesting that it represents an expression of small amounts of C56 rather than a new C-inhibitory activity. Thus, an individual with chronic nephritis lacking C7 is reported; the utility of a hemolytic screen assay in agarose plates for the detection of such patients is emphasized; persisting C56 is shown readily to be formed in this serum; and the presence of C7-consuming activity which is associated with and in all likelihood attributable to C56 is shown.

Structure and function of the pentraxins.

Over the past two years, the three-dimensional structure of the serum amyloid P component was defined by X-ray diffraction, the first such visualization of a pentraxin. Binding sites for calcium, ligands and complement were identified. New fusion proteins with amino acid sequence homology to the pentraxins were described, and new insights were gained into pentraxin phylogeny, biosynthesis, ligands, complement activation, leukocyte reactivity and biological functions in vivo.

Chemically shifted singlet oxygen spectrum.

The estimated light emission spectrum was determined for a singlet oxygen (1O2)-producing system, NaOCl + H2O2, alone and in the presence of tryptophan and bovine serum albumin. Tryptophan and bovine serum albumin caused a decrease in the red emission of 1O2 and an increase in the amount of shorter wavelength light. This effect was due to chemiluminescence rather than fluorescence. Arachidonic acid caused a similar spectral shift, while guanosine demonstrated a late chemiluminescent reaction of predominantly short wavelength light in the presence of 1O2.

Enhancement of human peripheral blood monocyte respiratory burst activity by aggregated C-reactive protein.

We had previously demonstrated that C-reactive protein (CRP), an acute phase reactant, when aggregated or coupled to a ligand, interacts with monocytes and certain human peripheral blood lymphocytes. The purpose of the present study, after further characterizing the binding interaction of CRP with human monocytes, was to focus on the biological response of monocytes to CRP binding. Flow cytometric analysis of human mononuclear leukocytes, following incubation with fluoresceinated heat-aggregated CRP (Agg-CRP), revealed that while greater than 70% of monocytes bound Agg-CRP, only 8% of lymphocytes demonstrated positive fluorescence. Furthermore, mean channel fluorescence values indicated that monocytes bound greater amounts of Agg-CRP per cell than did lymphocytes. Luminol-enhanced chemiluminescence (CL) was used as a measure of monocyte respiratory burst activity. Monocytes were stimulated only minimally by Agg-CRP alone; however, Agg-CRP substantially enhanced the CL response to heat-aggregated IgG. This Agg-CRP enhancing effect was selective for IgG-initiated monocyte activation, as no augmentation in CL was observed following cell stimulation with phorbol myristate acetate or serum-opsonized zymosan. These results demonstrate that aggregated CRP binds to the major proportion of human monocytes and selectively augments Fc receptor-mediated stimulation of monocyte oxidative metabolism.

In vivo decrease in the expression of complement receptor 2 on B-cells in HIV infection.

OBJECTIVE: To investigate changes in the expression of complement receptor 2 (CR2) on B-cells from HIV-infected individuals. CR2 is the C3d/Epstein-Barr virus receptor and has been implicated in B-cell activation. Changes in its level of expression may therefore be associated with B-cell dysfunction. DESIGN: Cross-sectional study of HIV-infected adults and age-matched control donors. METHODS: The percentage expression and mean fluorescence intensity of CR2 (and three additional markers: CD19, CD69, and a standard antigen designation: HLA-DR) was measured on CD20+ B-cells using a two-color flow cytometric assay. RESULTS: This study demonstrated a highly significant (P = 0.0001) decrease in the percentage co-expression of CR2 on CD20+ B-cells in HIV-infected individuals, compared with control donors. The mean percentage of CD20+ cells co-expressing CR2 was 71% (s.d., +/- 15%) in the HIV-seropositive patients and 94% (s.d., +/- 4%) in the control group. The pattern of CR2 expression in a number of the patients suggested a decrease in antigen density on the cells. Decreased expression of CR2 did not correlate with disease stage (asymptomatic, AIDS-related complex, or AIDS), nor with CD4+ T-cell percentage or absolute count, in the seropositive group. CONCLUSIONS: The evidence for a role for CR2 in B-cell activation suggests that its decreased expression, which we have demonstrated in HIV-seropositive individuals, may be associated with the B-cell dysfunction observed in HIV infection. Our finding that expression of this marker is decreased even in asymptomatic patients is consistent with reports of early B-cell defects in such individuals. Further investigation of this possible association may shed some light on both the increased incidence of bacterial infections in HIV-infected adults and children and their impaired responses to certain immunizations.

Complement and antibody mediate enhancement of HIV infection by increasing virus binding and provirus formation.

Previous studies have shown that infection of complement receptor (CR2)-bearing cells with HIV pretreated with antibody (Ab) plus complement (C) resulted in increased virus expression. The current study was designed to determine whether C-mediated 'enhancement' of HIV-1 production was the result of increased virus infection of cells as assessed by provirus formation and virus binding. Virus was incubated with anti-HIV Ab and/or C and added to CR2-positive MT-2 cells. Increased virus expression by MT-2 cells correlated with increased numbers of HIV-immunofluorescent-positive cells at 24 and 48 h and higher levels of provirus detected 8-28 h after infection. MT-2 cells also bound threefold more Ab-plus-C-treated virus than untreated virus. Serial dilutions of C showed that high levels of C with Ab did not enhance but rather suppressed virus expression. Studies were also performed which showed that terminal C components C5 and C8 were not necessary for the enhancing effect. The increased binding of C-coated HIV to CR-positive cells has important implications for the fate of virus in vivo.

The potential use of a dopamine neuron antibody and a striatal-derived neurotrophic factor as diagnostic markers in Parkinson's disease.

The cerebral spinal fluid (CSF) of patients with Parkinson's disease (PD) contains an antibody that immunocytochemically reacts with dopamine (DA) neurons in the substantia nigra (SN). This antibody was found in 78% of the CSF samples taken from patients with clinical PD. In contrast, only 3% of the CSF samples taken from control patients or patients with neurologic symptoms other than PD possessed this antibody. The production of this antibody might contribute to disease progression but does not appear to be the etiologic factor responsible for PD. In other experiments, concentrates of the CSF of patients with PD enhanced growth of mesencephalic cultures relative to control CSF. Both the antibody and the growth-promoting activity found in CSF are associated with degeneration of the SN and might therefore be useful as potential diagnostic markers for PD.

An enzyme-linked immunoabsorbent assay for the quantitation of the terminal complement complex from cell membranes or in activated human sera.

A sensitive and simple enzyme-linked immunoabsorbent assay (ELISA) has been developed to measure the terminal complement complex (TCC) in solution. Commercially available antibodies to the native complement (C) components C5 and C9 were used in a double antibody sandwich technique sensitive enough to detect 0.3 microgram/ml of purified TCC. The TCC was not detected in normal human serum (NHS) nor was it generated when sera from patients with a genetic deficiency of functional C5, C7, C8 beta or C9 were activated with cobra venom factor (CVF). If the C8 beta deficient serum was reconstituted with the C8 beta chain and incubated with CVF, TCC were formed and detected by the assay. In in vitro experiments, the TCC was detected in NHS activated by either the classical or alternative pathway even when there was no measurable consumption of C5, C8 or C9. In addition, adaptation of a detergent extraction procedure permitted the quantitation by the assay, of TCC which were generated on sensitized sheep erythrocyte membranes. Experiments to test sample handling conditions showed no generation of TCC in NHS after four freeze/thaw cycles and spontaneous formation only if NHS had been incubated at 37 degrees C for 48 h. The TCC in zymosan-activated NHS were stable at 37 degrees C for 1 week. Patients with C activation associated diseases such as SLE and rheumatoid arthritis had increased levels of TCC that correlated with positive clinical tests for inflammation, even though C levels were normal when measured by routine techniques. These results suggest that this ELISA will provide a valuable tool for studying the role of C in the pathogenesis of C-mediated diseases and in examining the mechanism of tissue injury in in vitro experimental systems.

Direct binding of complement component C1q to human immunodeficiency virus (HIV) and human T lymphotrophic virus-I (HTLV-I) coinfected cells.

Previous studies have shown that coinfection of the human T lymphotrophic virus type I (HTLV-I) chronically infected cell line MT4 with human immunodeficiency virus type 1 (HIV-1) results in cells which spontaneously activate complement via the classical pathway. This complement activation was antibody independent, yet required C2, suggesting either direct C1, C4, or C2 activation. Because some animal retroviruses have been shown to bind human C1q directly, the present study investigated the possible direct binding of C1q by HIV coinfected MT4 cells. Coinfected cells bound both C1q present in serum and highly purified C1q. Binding of C1q resulted in formation of active C1 on the cell surface, which could in turn activate complement as shown by C4 consumption. The C1q binding was not HIV-isolate specific since infection of MT4 cells with any of three diverse isolates all induced C1q binding. Purified collagen-like region (CLR) and globular region (GR) fragments of C1q both bound to coinfected cells, suggesting a mechanism of binding by C1q similar to that of fibronectin-C1q binding. However, culture of coinfected cells in serum-free (fibronectin-free) medium did not reduce C1q binding. A second HTLV-I chronically infected line, SLB-1, also displayed increased binding of C1q after HIV infection. The H9 cell line, which is not HTLV-I infected, did not bind C1q after HIV infection. These results suggest that a retrovirus protein expressed by coinfected cells directly binds C1q resulting in classical complement activation. This type of activation may have profound biological effects in persons coinfected with HIV-1 and HTLV-I.

Changes in respiratory burst activity during human monocyte differentiation in suspension culture.

Monocytes undergo a process of differentiation following their accumulation into extravascular spaces. This process has been examined previously by culturing monocytes and identifying changes in cell morphology, metabolism, and function over time. The present study was designed to characterize mononuclear phagocyte respiratory burst activity as related to differentiation by measuring chemiluminescence and superoxide anion generation in cultured human monocytes. Monocytes maintained in Teflon vials for up to 12 days increased in size, were positive for nonspecific esterase, and retained the ability to ingest latex particles. During culture, however, cells progressively lost their peroxidase-positive granules. When monocytes were cultured for one or five days, they elicited less than 50% of the luminol-enhanced chemiluminescence produced by fresh monocytes following PMA stimulation. By day 7, less than 20% of day 0 PMA-elicited chemiluminescence was observed. A comparable loss of serum-opsonized zymosan-induced chemiluminescence occurred during monocyte culture. Since it is recognized that luminol-enhanced chemiluminescence is, in large part, dependent upon myeloperoxidase and since differentiated mononuclear phagocytes are only minimally peroxidase-positive, cultured monocyte respiratory burst activity was also assessed by directly quantifying superoxide anion generation. When monocytes were cultured for three or five days, they elicited 38% more superoxide anion than did fresh monocytes following PMA stimulation. At day 7, PMA-induced superoxide anion release was comparable to day 0 levels. These data indicate that monocytes allowed to differentiate under nonadherent conditions maintain the ability to undergo a respiratory burst response as measured by superoxide anion release, but they concomitantly lose peroxidase-dependent luminol-enhanced chemiluminescence. In this regard, monocytes cultured in suspension metabolically resemble macrophages that have undergone differentiation within sites of inflammation.

Laboratory detection of complement activation and complement deficiencies.

The complement system is the major humoral amplification and effector mechanism of the immune system. Complement is activated in a variety of conditions, especially in immune-complex diseases. Activation can result in consumption of components involved in either the classical or the alternative complement pathway or both pathways. Such activation can be detected by laboratory analysis, quantitating specific complement component levels and measuring hemolytic function. An integrated approach to the laboratory evaluation of complement can ascertain which complement pathway is being activated, either in vivo or in vitro. Complement deficiency states are not rare, and a large enough number of such patients have now been detected for the patterns of disease susceptibility to be recognized. Since inherited deficiency of a complement protein may have dire consequences, it is important for laboratory technologists to recognize the patterns of reactivity associated with these deficiency states. The physician can then be alerted so that appropriate and effective preventive or therapeutic measures can be taken. In addition, the study of patients lacking an individual complement component has provided substantial understanding of the role of the complement system in host defense and inflammation.

The third component of complement (C3) bound to tumor target cells enhances their sensitivity to killing by activated macrophages.

The third component of complement (C3) bound to P815 tumor cells enhanced their susceptibility to killing by Corynebacterium parvum-activated murine macrophages (M phi). Hemolytically active normal mouse serum and C5-deficient mouse serum were used to deposit complement (C) on P815 tumor cells, in the absence of exogenous antibody, by an alternative pathway mechanism. Cell-bound C3 was detected and was quantified by using a cellular enzyme-linked immunospecific assay. Activated M phi produced tumor cytolysis in a serum-free 16-hr 51Cr-release assay. The lysis of C-treated tumor cells was increased over targets treated with sera containing 10 mM EDTA, heat-inactivated mouse sera, or medium. In addition, C alone did not cause specific 51Cr release. M phi elicited by casein or PBS did not lyse any of the tumor targets tested. The increase in lysis was dependent on the dilution of serum used, and was strongly correlated with the amount of C3 detected on the tumor cells. The enhanced lysis was abrogated by incubating C3-bearing tumor cells with F(ab')2 fragments of a goat anti-mouse C3 antibody. C treatment did not alter the kinetics of tumor cell lysis, nor did it enhance the binding of the targets to effector cells. These results suggest that C may regulate M phi-mediated killing of tumor cells by increasing the lytic efficiency of M phi that are in contact with target-bound C3.

Efficacy of HIV-specific and 'antibody-independent' mechanisms for complement activation by HIV-infected cells.

Previous studies in this laboratory have shown that efficient activation of complement (C) on HIV isolates and HIV-infected cells requires the binding of specific anti-HIV antibodies, while other investigators have observed 'antibody-independent' C activation. In an attempt to clarify these disparate findings, we investigated the effect of several variables on C activation by HIV-infected cells using flow cytometric analysis of C3 deposition. Antibody-mediated C activation using pooled sera from infected persons or human MoAbs directed against the V3 region of gp120 was always substantially higher than activation without antibody. Normal human serum (NHS) from a subset of HIV antibody-negative donors did, however, induce low levels of C3 deposition. Differences in C3 activation between the various NHS did not correlate with total haemolytic C levels or mannose-binding protein (MBP) levels. IgM isolated from NHS that induced high levels of C activation was at least partly responsible for the 'antibody-independent' C activation. Although there appeared to be a correlation between NHS that induced C activation and the presence of anti-blood type B IgM, absorption of anti-B did not abrogate the C3 deposition. Additionally, MoAb to the B antigen did not induce C3 deposition. These studies show that IgM in sera from HIV-uninfected donors can induce C3 deposition on HIV-infected cells, but that specific antibody-dependent C activation is substantially more efficient. Therefore, 'antibody-independent' C activation on HIV-infected cells may, in some cases, be more accurately described as HIV-cross-reactive antibody-dependent C activation.

Defined chemically cross-linked oligomers of human C-reactive protein: characterization and reactivity with the complement system.

Chemically cross-linked C-reactive protein (CRP) oligomers were prepared and characterized, and C1q binding and C activation were investigated. Purified human CRP was polymerized in the presence of both non-cleavable and cleavable cross-linking agents and further separated by Superose 12 analytical FPLC column chromatography into fractions of 110 KDa (pentameric monomers), 220 KDa (dimers) and 330 KDa (trimers); virtually no larger oligomers were formed under a variety of experimental conditions. CRP subunits were cross-linked both within and between CRP pentamers. CRP trimers retained native CRP antigenicity without expression of neo-CRP epitopes. CRP trimers showed maximal binding and CRP dimers showed partial binding of solid phase C1q while CRP monomers bound virtually no C1q at all; CRP trimers also bound to fluid phase C1q. Binding was Ca++ independent and increased as the ionic strength or pH were lowered, characteristics comparable to binding of aggregated IgG to C1q; it was not inhibited by phosphorylcholine. CRP trimers consumed total C, C1 and C2 haemolytic activities upon incubation in fresh human serum, but much less efficiently than did CRP-protamine complexes or Agg-IgG. CRP trimers failed to deplete alternative C pathway haemolytic activity at all. The stable, chemically defined CRP oligomers described in this report, which bind C1q efficiently but display poor ability to activate the classical C pathway in the absence of an appropriate ligand, should be valuable in further studies of the interactions between CRP and the C system.

Analysis of human C8 with monoclonal antibodies. Characterization of a monoclonal antibody that recognizes free C8 alpha-gamma subunit.

The eighth component of human C is essential for the formation of the membranolytic C attack complex. C8 has a unique structure in that two covalently linked chains, C8 alpha and C8 gamma, are associated non-covalently with the third chain, C8 beta. In order to study the structure and assembly of the C8 molecule, a panel of mAb has been produced against the C component C8. Eight of these mAb had reactivity to the C8 alpha-gamma subunit, whereas four reacted with C8 beta. One of the C8 alpha-gamma mAb, C8A2, had specificity for an epitope on the C8 alpha-chain and exhibited no cross-reactivity to any of the other terminal C components, including C8 beta. C8A2 inhibited the hemolytic activity of the C8 alpha-gamma subunit but had no effect on the activity of fluid phase whole C8 or C8 within membrane-bound C5b-8. Functional experiments suggest that C8A2 inhibits C8 alpha-gamma activity by interfering with its interaction with the C8 beta-chain. In an enzyme immunoassay using the C8A2 mAb, free C8 alpha-gamma subunit could be detected in both homozygous and heterozygous C8 beta-deficient serum. However, only low level binding was observed when homozygous C5- and C7-deficient sera were tested. Thus the mAb, C8A2, recognizes an epitope expressed on the C8 alpha-gamma subunit but not on intact C8 and can detect free C8 alpha-gamma in the presence of native C8.

Inherited deficiency of the ninth component of complement in man.

A 76-year-old man was found to have low (33% normal) serum complement (C) hemolytic activity, although C3 and C4 protein levels were normal. Further evaluation of his serum and plasma indicated that all C components were present in normal or elevated amounts except for C9, which was undetectable by both antigenic and functional assays. Addition of purified human C9 led to full restoration of the hemolytic activity. Family studies demonstrated that the deficiency was inherited as an autosomal codominant trait and was not linked with alleles at the HLA-A or HLA-B loci. The patient had no history of recurrent or unusual infections and no evidence of autoimmune disease. The availability of serum totally lacking in C9 permitted an investigation of the lytic capacity of the C5b-8 segment of the C attack mechanism, which was pursued in kinetic studies on the hemolysis of erythrocyte intermediates. These studies indicated that hemolysis occurred approximately 100 times slower in patient than in normal serum, using either EA or EAC1-7 intermediates as target cells. Serum bactericidal activity also was slower in patient serum, occurring at a rate about 1/35 that observed in normal serum. These studies provide direct independent evidence that cytolysis of erythrocytes and bacteria can be mediated by C5b-8, and allow a quantitative estimation of the increment in the rates of these reactions provided by normal serum levels of C9. The presence of readily detectable though slow hemolytic activity of C9-deficient serum may account for the difficulty in identifying individuals with this defect.