Incubation In Normal Human Serum Research Articles

Effects of two serine proteases from Bothrops pirajai snake venom on the complement system and the inflammatory response

The present study aimed to evaluate the effects of two serine proteases from Bothrops pirajai snake venom, named BpirSP27 and BpirSP41, on the complement system and the inflammatory response. The effects of these enzymes on the human complement system were assessed by kinetic hemolytic assays, evaluating the hemolysis promoted by the classical/lectin (CP/LP) and alternative (AP) pathways after incubation of normal human serum with the serine proteases. The results suggested that these enzymes were able to induce modulation of CP/LP and AP at different levels: BpirSP41 showed higher inhibitory effects on the hemolytic activity of CP/LP than BpirSP27, with inhibition values close to 40% and 20%, respectively, for the highest concentration assayed. Regarding AP, both enzymes showed percentages of inhibition of the hemolytic activity around 20% for the highest concentrations tested, indicating similar effects on this complement pathway. The proinflammatory effects of B. pirajai serine proteases were evaluated regarding their ability to induce paw edema, variations in the pain threshold and leukocyte recruitment at the site of injection. Both showed mild effects on these inflammatory processes, leading to low levels of increase of paw volumes and decrease in pain thresholds in rats up to 6 h after injection, and inducing neutrophil recruitment without significant increases in the total number of leukocytes in the inflammatory exudates after 6 and 24 h of administration into mice peritoneal cavity. These results suggest that serine proteases must present a minor role in the inflammation caused by B. pirajai snake venom.

Binding to complement factors and activation of the alternative pathway by Acanthamoeba

Acanthamoeba can cause severe ocular and cerebral diseases in healthy and immunocompromised individuals, respectively. Activation of complement appears to play an important role in host defence against infection. The exact mechanism, however, is still unclear. The aim of the present study was to investigate the effect of normal human serum (NHS) and normal mouse serum (NMS) on Acanthamoeba trophozoites, the binding of different complement factors to Acanthamoeba and the activation of the complement system. Moreover, we aimed to work out any possible differences between different strains of Acanthamoeba. A virulent T4 strain, a non-virulent T4 strain and a virulent T6 strain were included in the study. It was shown that NHS, but not NMS clearly has amoebicidal properties. After 5 min of incubation with NHS, amoebae showed plasma membrane disruption and extrusion of intracellular components. Cells were completely destroyed within 60 min of incubation in NHS but stayed intact after incubation in heat-inactivated serum. The binding of human C3 and C9 to amoebae was established by immunoblotting. Although incubation with mouse serum did not result in lysis of Acanthamoeba trophozoites an immunofluorescence assay (IFA) demonstrated a strong deposition of mouse complement factor C3 activation products, moderate binding of C1q, but no binding of MBL-A and MBL-C. EDTA inhibited the binding of C3 to acanthamoebae. Binding of amoebae to C3b was observed with sera from C1qa−/− and MBL-A/C−/− mice, but not with serum from Bf/C2−/− mice demonstrating an activation of complement via the alternative pathway. There were no significant differences between the three Acanthamoeba strains investigated. Altogether, our results prove that NHS is amoebolytic and that Acanthamoeba binds to C3 and C9 and activates the complement system via the alternative pathway.

Chylomicron accelerates C3 tick-over by regulating the role of factor H, leading to overproduction of acylation stimulating protein.

Acylation stimulating protein (ASP) is a fragment of the third component of complement (C3) that is generated in the presence of chylomicron, and plays a role in the synthesis of triacylglycerol by transporting free fatty acids into adipocytes. However, the precise mechanism of ASP generation, especially the role of chylomicron in ASP generation, is unknown. We examined the mechanism through which chylomicron induces ASP generation. Ultracentrifugationally separated chylomicron was incubated with normal human serum (NHS) under various conditions, and the amounts of complement activation products and ASP in the incubation mixture were determined by enzyme-linked immunosorbent assay (ELISA). Upon incubation of NHS with various amounts of chylomicron for 120 min, ASP was generated in a dose-dependent manner. The time course of the production of ASP was similar to the time course of the C3 tick-over phenomenon that occurred by depletion of factor H from the serum. The complement activation induced by chylomicron was different from the usual complement activation that occurs under the regulation of factor H and factor I with respect to the time course and the amount of ASP produced. Our results indicate that chylomicron accelerates C3 tick-over by regulating the role of factor H, leading to the overproduction of ASP.

Activation of the complement system and leukocyte recruitment by Tityus serrulatus scorpion venom

The scorpion Tityus serrulatus is considered one of the most dangerous species in Brazil. Its venom evokes an inflammatory response, although the exact mechanism of this effect is still unknown. The aim of the present study was to investigate the effect of Tityus serrulatus venom (TsV) on the complement system (CS) and on leukocyte recruitment. Complement consumption by TsV was evaluated using in vitro hemolytic assays, immunoelectrophoresis and two-dimensional immunoelectrophoresis of complement components (factor B and C3). In order to evaluate neutrophil migration induced in normal human serum (NHS) in the presence of TsV, in vitro chemotaxis assays were performed using the Boyden chamber model. In vitro TsV induced a concentration- and time-dependent reduction in hemolytic activity of the classical/lectin and alternative complement pathways, with samples of 43.0 μg and 43.4 μg, respectively, inhibiting 50% of the lytic activity. Alterations in C3 and factor B electrophoretic mobility after incubation of NHS with TsV, were identical to those obtained with zymosan (positive control). Incubation of NHS with TsV induced neutrophil chemotaxis similar to that observed with zymosan-activated serum. Our results show that TsV activates the CS, leading to factor B and C3 cleavage, to reduction of serum lytic activity and generation of complement chemotactic factors. Therefore, CS may play an important role in the inflammatory response observed upon scorpion envenomation.

Resistance of Acanthamoeba Species to Complement Lysis

Acanthamoeba species were evaluated for susceptibility to complement lysis as determined by release of radiolabeled uridine. The 3 Acanthamoeba species tested, A. culbertsoni (ATCC 30171), A. castellanii (ATCC 30010), and A. polyphaga (ATCC 30461), depleted hemolytic complement activity from normal human serum (NHS), yet were resistant to its lytic effects. Examination of microtiter plates containing amoebae incubated in NHS demonstrated formation of a pellet in the wells. Pellet formation was not observed when amoebae were incubated in human cord serum, heat-inactivated serum, or C1q-deficient serum. Ultrastructural examination of serum-treated amoebae revealed the presence of a finely granular substance that surrounded the amoebae. Treatment of amoebae with enzymes or metabolic inhibitors prior to incubation in NHS was performed to investigate the mechanism of complement resistance. Cycloheximide or cytochalasin D pretreatment increased the susceptibility of A. culbertsoni and A. castellanii to complement lysis. Cytochalasin D treatment also increased the susceptibility of A. polyphaga to complement lysis. Inhibition of serine protease activity by phenylmethylsulfonylfluoride increased complement susceptibility of all 3 species of Acanthamoeba. Enzymatic removal of surface components from A. polyphaga or A. castellanii, with trypsin, neuraminidase, or phosphatidylinositol-specific phospholipase C (PIPLC), did not affect serum resistance. In contrast, PIPLC treatment of A. culbertsoni significantly increased lysis by complement. The ability of Acanthamoeba species to activate the alternative complement pathway yet resist complement-mediated cellular lysis can be attributed to both the release of a transport-dependent extracellular matrix as well as the presence of complement inhibitory surface proteins.

Selection of Opa+ Neisseria gonorrhoeae by limited availability of normal human serum.

Experimental infections of human male volunteers with Neisseria gonorrhoeae have provided valuable insights into the early stages of gonorrheal disease. Bacterial variants expressing outer membrane opacity (Opa) proteins appear to be selected from the inoculum during a period in which total recoverable numbers of bacteria decrease rapidly. This apparent survival advantage occurs simultaneously with the onset of an inflammatory response, characterized by local production of interleukin 6 (IL-6) and IL-8 and the appearance of leukocytes in urine. Since the inflammatory response may also result in the presence of serum factors on the mucosal surface, we investigated the possibility that killing in normal human serum (NHS) leads to the selection of Opa+ variants. We therefore studied killing of separate populations and mixtures of Opa- and Opa+ N. gonorrhoeae MS11mk in NHS. Expression of an Opa protein conferred a survival advantage upon the organism; i.e., the Opa+ variants were more serum resistant than their isogenic Opa- counterparts, resulting in a selection for Opa+ phenotypes when a mixture of Opa+ and Opa- gonococci (GC) was exposed to submaximal doses of NHS. This selection was observed in three different lipooligosaccharide (LOS) backgrounds, indicating that it was not due to a difference in LOS expression between Opa- and Opa+ phenotypes. Incubation in NHS of sialylated GC resulted in a similar selection for Opa+ variants. The presence of normal human urine during the serum killing assay had no effect on the selection phenomenon but drastically depleted NHS of bactericidal activity, which was found to be at least partly due to complement inhibition. The results suggest that serum killing may contribute to the transition from Opa- to Opa+ phenotypes during the early stages of infection of the male urethra.

Membrane vesiculation of Naegleria fowleri amoebae as a mechanism for resisting complement damage.

Pathogenic and nonpathogenic Naegleria amoebae activate the alternative C pathway; however, only pathogenic amoebae are resistant to C-mediated damage. The present study was undertaken to determine the mechanism by which highly pathogenic N. fowleri amoebae resist C-mediated damage. Nomarski optics microscopy and electron microscopy of Naegleria amoebae revealed membrane blebbing on the surface of C-resistant N. fowleri, but not on C-sensitive N. gruberi, in response to incubation in normal human serum diluted 1:4 to 1:16. Immunofluorescent staining of pathogenic amoebae, by using antiserum to human C proteins comprising the membrane attack complex, C5b through C9, and FITC-labeled goat anti-rabbit IgG, confirmed that the membrane attack complex was concentrated on the membrane blebs. Binding studies with the use of radioiodinated C9 demonstrated a decrease in the 125I-labeled C9 cpm associated with N. fowleri amoebae and an increase in the 125I-labeled C9 cpm associated with the released membrane vesicles after increasing incubation periods in normal human serum. Treatment of pathogenic, C-resistant N. fowleri with cytochalasin D or cytochalasin B to inhibit actin-dependent exocytic processes increased the susceptibility of the amoebae to C damage. In contrast, incubation of nonpathogenic, C-sensitive amoebae with cytochalasins did not alter their susceptibility to C lysis. These data indicate that pathogenic N. fowleri use membrane vesiculation to remove membrane-deposited C proteins, specifically the membrane attack complex (C5b-C9). The ability to remove surface-associated membrane attack complexes serves as one mechanism by which pathogenic N. fowleri resist C lysis.

Accelerated decay of C3b to iC3b when C3b is bound to the Cryptococcus neoformans capsule.

Incubation of encapsulated and nonencapsulated Cryptococcus neoformans in normal human serum (NHS) leads to activation and binding of potentially opsonic fragments of complement component C3 to the yeast cells. Analysis of the molecular forms of C3 after incubation of encapsulated cryptococci in NHS showed that the percentage of bound C3 occurring as iC3b approached 100% after 8 min. The percentage of bound C3 occurring as iC3b on nonencapsulated cryptococci never exceeded 70%, even after 60 min of incubation in NHS. Conversion of C3b to iC3b was assessed further by incubating C3b-coated cryptococci for various times with a mixture of complement factors H and I at 40% of their respective physiological concentrations. Most, if not all, of the C3b on encapsulated cryptococci was converted to iC3b at a single fast rate. Conversion of C3b to iC3b on nonencapsulated cryptococci did not follow a single rate constant and appeared to have a fast and a slow component. Studies of the requirements for factors H and I in cleavage of C3b to iC3b showed steep dose-response curves for both factors in the case of encapsulated cryptococci and shallow curves with C3b bound to nonencapsulated cryptococci. Taken together, our results indicate that C3b molecules bound to encapsulated cryptococci have a uniformly high susceptibility to conversion to iC3b by factors H and I. In contrast, a significant portion of the C3b bound to nonencapsulated cryptococci is very resistant to conversion to iC3b by factors H and I.

Binding and release of C3 from Leishmania donovani promastigotes during incubation in normal human serum.

We have examined the nature and extent of C3 deposition on Leishmania donovani, strain 1S, clone 2D, promastigotes. Total molecules of C3 bound/parasite after 60 min was similar for parasites incubated in normal human serum, normal human serum adsorbed to remove natural antibody, or either serum source chelated with Mg-EGTA to limit activation to the alternative pathway. A comparison of parasites grown to early, mid, late-log or stationary phases revealed no difference in the extent and kinetics of C3 binding. C3 bound covalently to the parasite primarily through a hydroxylamine resistant (putatively amide) linkage. Of the bound C3, 75% was present as hemolytically inactive iC3b. Nearly 50% of the bound C3 was spontaneously released within 30 min at 37 degrees C. This spontaneous release was due to an unusual proteolytic cleavage event that released C3 from the C3 acceptor on the parasite surface. These results define and characterize the unusual features of C3 binding to L. donovani promastigotes during incubation in serum.

Resistance of highly pathogenic Naegleria fowleri amoebae to complement-mediated lysis.

Weakly pathogenic and nonpathogenic Naegleria spp. are readily lysed by human and guinea pig complement. Highly pathogenic Naegleria fowleri are resistant to complement-mediated lysis. Electrophoretic analysis of normal human serum (NHS) incubated with pathogenic or nonpathogenic Naegleria spp. demonstrates that amoebae activate the complement cascade, resulting in the production of C3 and C5 complement cleavage products. To determine whether surface constituents play a role in resistance to complement lysis, trophozoites of Naegleria spp. were subjected to enzymatic treatments prior to incubation in NHS. Treatment of trophozoites with papain or trypsin for 1 h, but not with neuraminidase, increased susceptibility of highly pathogenic Naegleria fowleri to complement lysis. Treatment of trophozoites with actinomycin D or cycloheximide during incubation with NHS or pretreatment with various protease inhibitors for 4 h did not increase the susceptibility of N. fowleri amoebae to lysis. Neither a repair process involving de novo protein synthesis nor a complement-inactivating protease appears to account for the increased resistance of N. fowleri amoebae to complement-mediated lysis.

Complement component C1q enhances invasion of human mononuclear phagocytes and fibroblasts by Trypanosoma cruzi trypomastigotes.

Internalization and infectivity of Trypanosoma cruzi trypomastigotes by macrophages is enhanced by prior treatment of parasites with normal human serum. Heating serum or removing C1q from serum abrogates the enhancement, but augmentation of attachment and infectivity is restored by addition of purified C1q to either serum source. Although both noninfective epimastigotes (Epi) and vertebrate-stage tissue culture trypomastigotes (TCT) bind C1q in saturable fashion at 4 degrees C, internalization by monocytes and macrophages of TCT but not Epi-bearing C1q is enhanced in comparison to untreated parasites. Adherence of human monocytes and macrophages to surfaces coated with C1q also induces a marked enhancement of the internalization of native TCT. C1q enhances attachment of both Epi and TCT to human foreskin fibroblasts, but only when C1q is on the parasite and not when the fibroblasts are plated on C1q-coated surfaces. Only TCT coated with C1q show enhanced invasion into fibroblasts. Although trypomastigotes produce an inhibitor of the complement cascade which limits C3 deposition during incubation in normal human serum, C1q binds to the parasite and enhances entry of trypomastigotes into target cells.

Effect of membrane phospholipids on activation of the alternative complement pathway.

Although some of the membrane glycoproteins that serve as activators or regulators of C activation have been identified, the influence of membrane lipids has not been studied extensively. A model of alternative C pathway activation was established using liposomes composed of cholesterol and synthetic phospholipids. Liposomes containing phosphatidylcholine (PC) as the sole phospholipid did not activate C as measured by C3 binding after incubation in normal human serum containing 2.5 mM MgCl2 and 10 mM EGTA. When phosphatidylethanolamine (PE) was included as 20% or more of the phospholipid, C3 binding was observed. C3 binding to liposomes was inhibited by salicylhydroxamic acid indicating binding through the C3 thioester bond. The phospholipid composition did not influence C3 binding to liposomes in an unregulated system of C3, B, D, and P indicating equivalent C3b binding sites on activating and nonactivating liposomes. When the regulatory proteins H and I were added to the other components, liposomes containing PE bound three times more C3 than PC liposomes suggesting that the phospholipid affects C3 regulation. This was tested directly in a radiolabeled H binding assay. In the presence of equal amounts of C3b, PC liposomes showed a greater number of high affinity H binding sites than PE liposomes. Using different PE derivatives, C activation could be directly related to the phospholipid polar head group. Liposomes containing PE, trinitrophenyl-PE or monomethyl-PE did activate the alternative C pathway, whereas those containing dimethyl-PE, PC, or phosphatidylserine did not. These studies provide evidence that primary and secondary amino groups on lipid membranes can decrease the interaction between H and C3b and provide sites for alternative pathway activation.

血液透析による補体系の変動―補体分解産物及びTerminal complement complexの検討

Incubation of normal human serum with hemodialysis membranes in vitro resulted in conversion of C3 which was detected by crossed immunoelectrophoresis. In addition, formation of C3d was also observed, as was detected by double-decker rocket immunoelectrophoresis. Furthermore, breakdown products of complement (iC3b, C4d & Bb) in plasma samples were measured by ELISA. The microassay plates were coated with monoclonal antibodies which bind specifically to human iC3b, C4d and Bb, respectively, while the plasma samples were drawn from five polycystic kidney patients during initial hemodialysis. As a result, the iC3b and Bb levels in plasma were seen to increase during hemodialysis but the C4d levels revealed no significant changes. It was also observed that the Bb levels in patients undergoing hemodialysis were significantly higher than those in various renal and collagen diseases. Terminal complement complex (TCC) was not present in detectable amounts in normal human serum when measured by ELISA. However, incubation of normal human serum with hemodialysis membranes in vitro resulted in detection of TCC. TCC was present in the same plasma samples drawned from the five polycystic kidney patients during initial hemodialysis. It is suggestive that activation of complement by hemodialysis membrane is predominantly mediated through the alternative pathway and products such as TCC and anaphylatoxin are formed by this activation.

Strain variation in phagocytosis of Cryptococcus neoformans: dissociation of susceptibility to phagocytosis from activation and binding of opsonic fragments of C3.

Phagocytosis of Cryptococcus neoformans is markedly influenced by the presence of a polysaccharide capsule. We examined activation and binding of C3 fragments to eight isolates of C. neoformans. All isolates were shown to have capsules by light and electron microscopy. These strains differed in susceptibility to phagocytosis by neutrophils. Yeast cells were opsonized by incubation in normal human serum. Five strains were resistant to ingestion, two strains showed intermediate levels of resistance to ingestion, and one strain was quite sensitive to phagocytosis. Yeast cells opsonized with heat-inactivated serum (56 degrees C for 30 min) neither attached nor were ingested by neutrophils. A quantitative estimate of the amount of C3 bound to the yeast cells was determined by use of normal human serum containing 125I-labeled C3. The results showed approximately 5 X 10(6) to 10 X 10(6) C3 molecules per yeast cell regardless of whether the yeast cells were sensitive or resistant to phagocytosis. Bound C3 was eluted from the yeast cells by treatment with 0.1 M NH2OH (pH 10), and the eluted fragments were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Results of this analysis showed that little of the C3 was in the form of C3b, and there was substantial decay to iC3b, the inactive decay product of C3b. This pattern of decay was similar with all strains. Immunoelectron microscopy was used to assess the ultrastructural location of the C3 fragments bound to the yeast cells. C3 fragments were bound to the perimeter of the capsule regardless of whether the isolate was sensitive or resistant to phagocytosis. Thus, phagocytosis-sensitive and phagocytosis-resistant isolates were similar with regard to the amount, molecular form, and ultrastructural location of C3 fragments bound to the cryptococcal capsule. These results further indicate that activation of the complement cascade is necessary but not sufficient for phagocytosis of the yeast cell.

Fibronectin binding to C1q associated with antigen-antibody complexes in EDTA-treated plasma.

In this report we have investigated the association of fibronectin with antigen-antibody-C1q complexes incubated in fibronectin-depleted and C1q-depleted plasma. When BSA--anti-BSA immune aggregates are incubated in plasma depleted of both fibronectin and C1q to which 125I-fibronectin has been reconstituted, little radio-activity is bound to the immune complexes. However, pre-incubation of immune complexes with purified C1q prior to incubation in the plasma causes an approximately 10-fold increase in the amount of radioactivity bound. The binding of 125I-fibronectin to preformed antigen-antibody-C1q complexes is specific, since the reaction is inhibited by the addition of unlabelled fibronectin but not by ovalbumin. When antigen-antibody-C1q complexes are incubated in C1q-depleted plasma containing physiological concentrations of fibronectin, and analysed by immunoblotting, fibronectin antigens are detected on the immune complexes. Identical results are obtained using immune complexes composed of sheep erythrocyte rabbit anti-sheep erythrocyte C1q (EAC1q) cells. There is no specific requirement for preformed antigen-antibody-C1q complexes, since fibronectin can be detected on antigen-antibody complexes after incubation in normal human serum or in C1q-depleted ethylenediamineteraacetic acid (EDTA) serum reconstituted with purified C1q prior to incubation with the complexes. Finally, we also demonstrate that in the presence of C1q, 125I-fibronectin will associate with soluble antigen-antibody complexes.

Activation of the alternative pathway of complement by DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (Mergetpa).

Incubation of normal human serum with 3-5 mM Mergetpa causes activation of the alternative pathway of complement as indicated by fragmentation of C3 and factor B, dependent on the presence of divalent cations. Mergetpa inhibits the regulatory protein factor I and, as a consequence, may allow initiation of the feedback cycle of the alternative pathway by deregulation. The classical pathway is not or only to a minor extent activated. A decrease in the hemolytic activity of C2, C5 and one or several of the components C6-9 in the presence of 5 mM Mergetpa may be explained by direct inactivation. The described effects are probably mediated by Mergetpa acting as a thiol.

A circulating inhibitor of fluid-phase amplification. C3 convertase formation in systemic lupus erythematosus.

C3 nephritic factor (C3NeF) was used to assess the formation of the fluid-phase amplification convertase, C3b,Bb, in 37 serum specimens from 24 patients with systemic lupus erythematosus (SLE). C3b,Bb formation was measured by the concentration of Ba, released when C3b,B is activated. Incubation of normal human serum (NHS) with C3NeF accelerates C3b amplification loop turnover with the formation of large quantities of C3b,Bb. In contrast, sera from 22 of 24 patients with SLE formed little or no convertase when incubated with C3NeF. C3 conversion to C3b was commensurately reduced. The inhibition could not be attributed to depressed serum concentrations of C3, factor B, or classical pathway components. Inhibitor present in excess could be demonstrated in 23 of 34 specimens of SLE serum by mixing experiments. The spontaneous convertase formation that occurs when a portion of the serum H is inactivated with F(ab')2 anti-H was also shown to be inhibited in SLE serum. The inhibition was found, however, to be H dependent in that convertase formation was normal in SLE serum depleted of H. It is concluded that the C3b in most SLE sera is unusually susceptible to inactivation by H, but a functional abnormality was not demonstrable in either C3 or H isolated from SLE serum. The inhibition could be simulated in NHS by addition of heparin, 100 micrograms/ml. In vivo, inhibition of convertase formation could interfere with the solubilization and disposal of immune complexes by reducing the deposition of C3b on the immune complex lattice.

Effect of different sulfonamides on the human serum complement system.

Incubation of normal human serum with different sulfonamides led to a dose-dependent inactivation of total hemolytic complement activity. A decrease of the activities of C1, C2, C3, C5 and one or several of the components C6-9 was observed after treatment of normal human serum with the sulfonamide sulfisomidine. Inactivation of complement components by sulfonamides seems to result from direct interaction with the drug as well as, to a minor extent, from activation of the alternative pathway. At relatively high concentrations, sulfonamides caused a conformational change in C3 and C4. The observed structural change is equivalent to that induced by cleavage of the internal thiolester bond in these molecules. The generation of such structurally altered C3 (C3b-like C3) as well as an antagonizing effect of sulfonamides towards the action of the regulatory protein factor I might be responsible for alternative pathway activation. The physiological relevance in vivo of the observed effects of sulfonamides remains to be assessed.

Mediation of the killing of rough, mucoid isolates of Pseudomonas aeruginosa from patients with cystic fibrosis by the alternative pathway of complement.

The mechanism of killing of 12 serum-sensitive strains of mucoid Pseudomonas aeruginosa isolated from patients with cystic fibrosis was investigated. A quantitative assay indicated that more than 90% of cells were killed in 50% normal human serum (NHS). All strains failed to grow in NHS concentrations of greater than 10%. Killing was unaffected by adsorption of NHS with the mucoid bacteria or chelation with MgCl2-ethyleneglycol bis(beta-aminoethyl ether)N,N1-tetraacetate (MgCl2-EGTA) but was abolished in serum heated to 50 C for 20 min. Incubation of NHS with mucoid P. aeruginosa reduced the hemolytic capability of MgCl2-EGTA-chelated NHS against rabbit red blood cells by 56%-99%. Killing of the serum-sensitive mucoid strains was also seen in hypogammaglobulinemic serum. These data suggest that killing of such strains by NHS can occur via antibody-independent activation of the alternative pathway of complement. The importance of this finding lies in the implication that complement levels in the lungs of patients with cystic fibrosis who are colonized by these organisms are inadequate to deal with this chronic, progressive infection.

Reduction of Phagocyte Adherence by Nephritic Sera: Relation to Complement Activation

Phagocytes isolated from either normal donors or from patients with poststreptococcal (P-SGN), lupus erythematosus (SLE-GN), or membranoproliferative (MPGN) glomerulonephritis showed normal adherence to glass (PAg) after incubation in normal human serum (NHS), but was reduced after incubation in patient serum. Low PAg was the consequence of incubation of normal phagocytes with the earliest available sera from all 22 P-SGN patients, 28 of 37 SLE-GN patients, 19 of 25 patients with MPGN type I, all 10 with types II and III, and all 5 with nephritis associated with chronic bacteremia. Low C3 and decreased PAg were related by regression analysis in sera from patients with P-SGN (P less than 0.001), SLE-GN (P less than 0.005), and MPGN (P less than 0.001) type I. In patients with P-SGN and one patient with nephritis associated with chronic bacteremia, complement levels and PAg returned to normal in parallel with clinical improvement. In vitro, PAg was reduced by NHS treated with either zymosan or bovine serum albumin (BSA)-anti-BSA complexes but neither BSA-anti-BSA complexes or zymosan, previously incubated in NHS, reduced PAg. PAg was normal in serum deficient in C4 or C5 unless treated with zymosan.