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Atypical hemolytic uremic syndrome is an ultra-rare disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute kidney injury. Its pathogenesis is driven most frequently by dysregulated cell-surface control of the alternative pathway of complement secondary to inherited and/or acquired factors. Here we evaluated two unrelated patients with atypical hemolytic uremic syndrome. The first, a five-year-old Caucasian female, presented at 10 months with schistocytes, thrombocytopenia and kidney injury. The second, a 55-year-old Caucasian female, presented at age 31 following caesarean section for preeclampsia. Complement biomarker testing was remarkable for undetectable levels of C3 in both. Circulating levels of C5 and properdin were also low consistent with over-activity of the alternative and terminal pathways of complement. Genetic testing identified a heterozygous novel variant in CFB (c.1101 C>A, p.Ser367Arg) in both patients. Functional studies found strong fluid-phase C3 cleavage when normal and proband sera were mixed. Cell-surface C3b deposition was strongly positive when patient serum was supplemented with C3. In vitro control of C3 convertase activity could be restored with increased concentrations of factor H. Thus, CFB p.Ser367Arg is a gain-of-function pathogenic variant that leads to dysregulation of the alternative pathway in the fluid-phase and increased C3b deposition on cell surfaces. Our study highlights the complexities of complement-mediated diseases like atypical hemolytic uremic syndrome and illustrates the importance of functional studies at the variant level to gain insight into the disease phenotype.

The complement cascade is an ancient defense system that destroys and eliminates threats to normal homeostasis in the bloodstream and tissues. Although multiple controls keep complement in check to minimize innocent bystander injury to normal cells and tissues, defects in complement regulation due to mutations in, or autoantibodies to, complement control proteins underlie the pathogenesis of several hemolytic diseases including paroxysmal nocturnal hemoglobinuria, and atypical hemolytic uremic syndrome. In autoimmune hemolytic anemias complement plays an important role in erythrocyte destruction mediated by antierythrocyte antibodies. The pathogenic mechanisms of these hemolytic diseases are discussed, with an emphasis on pivotal steps in complement activation.

This study hypothesized that microvascular retention of phosphatidylserine-containing microbubbles (MB-PS) would allow detection of recent but resolved myocardial ischemia with myocardial contrast echocardiographic (MCE) molecular imaging.Techniques for ischemic memory imaging which can detect and spatially assess resolved myocardial ischemia are being developed for rapid evaluation of patients with chest pain.MCE molecular imaging with MB-PS was performed 1.5 h, 3.0 h, and 6.0 h after brief (10 min) myocardial ischemia in mice; data were compared to selectin-targeted microbubbles. MCE molecular imaging with Sonazoid (GE Healthcare, Amersham, United Kingdom), a commercially produced phosphatidylserine (PS) - containing agent, was performed in separate mice at 1.5 h and 3.0 h after ischemia-reperfusion; and in dogs undergoing 135 min of ischemia and 60 min of reflow as well as in closed-chest nonischemic control dogs. The mechanism for MB-PS attachment was assessed by intravital microscopy of post-ischemic muscle and by flow cytometry analysis of cell-MB interactions.In mice undergoing ischemia-reperfusion without infarction, signal enhancement in the risk area for MB-PS and p-selectin glycoprotein ligand-1-targeted microbubbles was similar at reflow times of 1.5 h (23.3 ± 7.3 IU vs. 30.7 ± 4.1 IU), 3.0 h (42.2 ± 6.2 IU vs. 33.9 ± 7.4 IU), and 6.0 h (24.1 ± 4.3 IU vs. 25.5 ± 4.7 IU). For both agents, signal in the risk area was significantly (p 0.05) higher than remote region at all reflow times. Sonazoid also produced strong risk area enhancement at 1.5 h (34.7 ± 5.0 IU) and 3.0 h (52.5 ± 4.5 IU) which was approximately 3-fold greater than in the control region, and which correlated spatially with the microsphere-derived risk area. In dogs, Sonazoid signal in the risk area was5-fold higher than in closed-chest control myocardium (42.2 ± 8.1 IU vs. 7.9 ± 3.3 IU; p 0.001). Mechanistic studies indicated that MB-PS attached directly to venular endothelium and adherent leukocytes which was dependent on serum complement components C1q and C3.Ischemic memory imaging with MCE is possible using MB-PS which may obviate the need for ligand-directed targeting.

Immune complexes formed between monoclonal antibodies (mAbs) and toxins can neutralize toxicity in vivo by multiple mechanisms. Toxin sequestration and clearance by mAbs may be improved by enhancing their ability to bind to red blood cells (RBCs) through immune adherence. This can be achieved by converting the mAbs to heteropolymers (HPs), which are antigen-specific mAbs cross-linked to mAbs targeting the complement receptor (CR1), a protein that is expressed on the surface of RBCs in primates and mediates delivery of complement C3b-containing immune complexes to tissue macrophages. Conversion of mAbs to HPs has been shown to enhance clearance of multivalent antigens from the blood circulation, but the interaction of HPs with monovalent toxins has not been examined. Using botulinum neurotoxin (BoNT) as a model system, we studied the effect of conversion of a pair of BoNT-specific mAbs into HPs on toxin neutralization and handling in vivo. Two HPs given in combination had 166-fold greater potency than un-modified mAbs, neutralizing 5,000 LD50 BoNT, when tested in transgenic mice expressing human CR1 on RBC membranes. Improvement required adherence of BoNT to the RBC in vivo and 2 HPs, rather than an HP + mAb pair. The HP pair bound BoNT to RBCs in the circulation for 2 hours, in comparison to BoNT-neutralizing anti-serum, which induced no detectable RBC binding. HP pairs exhibited enhanced uptake by peritoneal macrophages in vitro, compared to pairs of mAbs or mAb + HP pairs. In a post-exposure therapeutic model, HPs gave complete protection from a lethal BoNT dose up to 3 hours after toxin exposure. In a pre-exposure prophylaxis model, mice given HP up to 5 days prior to BoNT administration were fully protected from a lethal BoNT dose. These studies elucidate general mechanisms for the neutralization of toxins by HP pairs and demonstrate the potential utility of HPs as BoNT therapeutics.

β2-Microglobulin (β2M), the light chain of the class I major histocompatibilty complex (MHC-I), is a promising tumor target for monoclonal antibodies (mAbs) in cancer immunotherapy. Several reports indicate that chelation of cell-associated β2M by specific mouse mAbs promotes tumor cell destruction by inducing apoptosis or other cytotoxic signaling pathways. Human mAbs employed in cancer therapy are usually IgG1, which mediates cell-killing by effector mechanisms including complement dependent cytotoxicity (CDC). The analogous mouse IgG2a and IgG2b isotypes are similarly effective in activating complement. Therefore, we examined the complement-activating properties of anti-β2M mouse mAbs 1B749 (IgG2a) and HB28 (IgG2b) when either mAb was bound to tumor cell lines or normal cells; we compared these β2M-specific mAbs with mouse mAb W6/32 (IgG2a), specific for human leukocyte antigens in the MHC-I heavy chain. All three mAbs bind to most human cell lines and normal cells in approximately equal amounts, consistent with a 1:1 stoichiometry for the HLA heavy chain in association with β2M. The three mAbs promote rapid C3b deposition and substantial CDC of human cell lines, and mAbs 1B749 and W6/32 have robust cytotoxic activity on reaction with normal mononuclear cells and platelets. Curiously, mAb HB28 induces modest C3b deposition and little CDC of normal cells, and its weaker complement-fixing activity was confirmed by ELISA. Based on these findings, we suggest that human IgG mAbs that target β2M for cancer immunotherapy be selected or engineered so as not to activate complement, thus eliminating the potential adverse effects of complement-mediated lysis of normal cells.

The anti-CD20, B-cell-specific mAb rituximab (RTX) has been approved for treatment of non-Hodgkin's B cell lymphoma and rheumatoid arthritis. Under conditions of high B cell burden, exhaustion of the body's effector mechanisms, for example, NK-cell-mediated killing, may lead to substantial decreases in the immunotherapeutic efficacy of this mAb. Moreover, RTX treatment of patients with chronic lymphocytic leukemia and high levels of circulating B cells can lead to removal of CD20 from the cells, thus allowing them to persist and resist clearance. RTX therapy for several autoimmune diseases has proven to be effective, but in numerous instances there has been little correlation between reductions in disease activity and changes in titers of pathogenic autoantibodies. This paradox may be explained by a separate mechanism: Binding of RTX to B cells generates immune complexes that act as decoys to attract monoycte/macrophages and thus reduce their inflammatory activity in certain autoantibody-mediated diseases. Several second-generation anti-CD20 mAbs with enhanced cytotoxic action have been developed and are being tested in the clinic for treatment of cancer and autoimmune diseases. The application of these mAbs, potentially in combination with immune effector modifying drugs, may successfully address the shortcomings of current anti-CD20 immunotherapy.

Binding of the chimeric, humanized anti-CD20 mAb Rituximab (RTX) to B lymphocytes activates complement and promotes covalent deposition of C3 fragments (C3b/iC3b) on cells. Previous fluorescence microscopy studies, based on examination of B cell lines and of blood samples from RTX-treated CLL patients, suggest that C3b/iC3b is closely associated with cell-bound RTX. We examined Raji cells opsonized with serum and RTX with the ImageStream imaging flow cytometer. Cells were stained with fluorescently-labeled RTX and mAbs specific for C3b/iC3b fragments or for human IgG, and then imaged using the ImageStream cytometer and analyzed with an algorithm (Similarity Bright Detail Score, SBDS) which tests for co-localization of fluorescent probes. SBDS, calculated on 10,000 cells, verified that the majority of deposited C3b/iC3b is co-localized with bound RTX. In contrast, when cells were first opsonized in serum alone, washed and then reacted with RTX, SBDS confirmed that RTX and C3b/iC3b are poorly co-localized, thus demonstrating that cell-bound RTX directs deposition of C3b. In addition, a sulfhydryl-specific probe, maleimide conjugated to AF488, exhibited substantial co-localization with an anti-C3b/iC3b mAb on Raji cells opsonized with RTX and serum, thus validating maleimide labeling as an alternative for detecting cell-bound C3b/iC3b. The digital imaging method described should have wide applicability for quantitative analysis of co-localization.

The alternative pathway (AP) of the complement system plays an important role in tissue damage and inflammation associated with certain autoimmune diseases and with ischemia-reperfusion injury. Selective inhibition of the AP could prevent such pathologies while allowing the classical and lectin pathways of complement activation to continue to provide protection. Here we present data describing selective inhibition of the AP of complement by anti-C3b/iC3b monoclonal antibody (mAb) 3E7, and by a chimeric, "deimmunized" form of this mAb, H17, which contains the human IgG1 Fc region and was further modified by substitution of amino acids in order to remove T cell epitopes. Both mAbs block AP-mediated deposition of C3b onto zymosan or Sepharose 4B, and they also inhibit AP-promoted lysis of rabbit erythrocytes. MAbs 3E7 and H17 also successfully compete with both factors B and H for binding to C3b-opsonized substrates, and the ability of both mAbs to inhibit the AP is blocked by pre-incubation with two different sources of C3(H2O). Kinetic measurements demonstrate that mAb 3E7 effectively stops progression of C3b deposition after AP activation is initiated. Our results therefore suggest that these mAbs block activation of the AP by binding to both C3(H2O) and to C3b, and thus prevent binding and activation of factor B. Based on these and other observations, mAb H17 may find future use in therapeutic applications focused on selective inhibition of the AP.

Rituximab (RTX) is a monoclonal antibody which targets CD20 and is approved for treatment of non-Hodgkin's lymphoma (NHL), with an approximate 50% overall response rate among NHL patients. Accurate determination of RTX concentrations in patient plasmas is important for proper dosing of patients and for correlating RTX concentrations with clinical responses. There is currently no assay available for RTX which utilizes easily obtainable commercial reagents. Therefore, we sought to develop such an assay, and in this report we describe three new assays for RTX concentration. One assay, based on flow cytometry, quantitates immunologically active RTX based on its ability to bind to CD20 on Raji cells. Two other methods, based on flow cytometry and ELISA, measure RTX based on its antigenic properties. The assays are accurate, in good agreement with one another, and can all measure RTX concentrations as low as approximately 1 microg/ml in both sera and plasmas. Use of these assays reveals that chronic lymphocytic leukemia (CLL) patients receiving RTX treatment have lower plasma RTX concentrations than patients with other B cell lymphomas at all times over the usual 4-week course of therapy. The level in CLL plasmas often declines to

ObjectiveThe goal of the study was to determine whether microbubble charge influences the microvascular retention of microbubble contrast agents.BackgroundInteractions between serum proteins and lipid membranes are greater with anionic compared with neutral membranes. These interactions may influence the microvascular behavior of anionic lipid microbubbles.MethodsIntravital microscopy of the cremaster muscle was performed in six wild-type mice and three C3-deficient mice during intravenous injection of lipid-shelled microbubbles with either a neutral or a negative charge. Both agents were prepared with and without a protective surface layer of polyethyleneglycol (PEG). Complement attachment to microbubbles was assessed by flow cytometry with flourescein isothiocyanate-conjugated anti-C3b monoclonal antibody. Myocardial contrast echocardiography was performed in six dogs to assess pulmonary and myocardial retention of microbubbles.ResultsSize-independent capillary retention of microbubbles, occurring for a few seconds to >10 min, was frequently observed with anionic, but rarely with neutral, microbubbles (4.3 ± 0.3 vs. 0.4 ± 0.1 mm−3, p < 0.01). Anionic microbubble retention was reduced by 70% by surface PEG and was also markedly reduced in C3-deficient mice (1.4 ± 0.1 mm−3, p < 0.05 vs. wild-type). Flow cytometry demonstrated complement attachment to only anionic microbubbles. Contrast echocardiography indicated both pulmonary and myocardial retention of only anionic microbubbles, the latter evidenced by persistent opacification >10 min after bolus intravenous injection.ConclusionsLipid microbubbles with a net negative charge can be retained within capillaries via complement-mediated attachment to endothelium. This property may be useful for the development of ultrasound contrast agents that can be imaged late after venous injection.

Immune complexes (IC) bound to the primate erythrocyte (E) complement receptor (CR1) are cleared from the circulation of primates and localized to phagocytic cells in the liver and spleen without E destruction. IC can be bound to E CR1 either via C3b opsonization or with cross-linked mAb complexes (heteropolymers, HP) which contain a mAb specific for CR1 and a mAb specific for an antigen. The long-term goal of our work is to apply the HP system to the treatment of human diseases associated with blood-borne pathogens. This review discusses the mechanism by which the E-bound IC are transferred to acceptor cells. Our studies in animal models as well as our in vitro investigations indicate that IC transfer is rapid (usually >90% in 10 min) and does not lead to lysis or phagocytosis of the E. Experiments with specific inhibitors and the use of IC prepared with Fab′ fragments suggest that transfer depends mainly upon recognition by Fc receptors on the acceptor cell. Moreover, we find that IC release from the E is associated with a concerted loss of CR1, and is followed by uptake and internalization of the IC by the acceptor cell. We suggest that recognition and binding of the E-bound IC substrates by Fc receptors allows close contact between the E and acceptor cells, which in turn facilitates proteolysis of E CR1, presumably by a macrophage-associated protease. After proteolysis, the released IC are internalized by the macrophages.

Red blood cells (RBC) modified with biotin and streptavidin (SA) present an interesting potential drug delivery system. Biotinylation and SA attachment, however, alter the biocompatibility of RBC. We have reported that polyvalent SA attachment induces lysis of biotinylated RBC (b-RBC) by homologous complement via the alternative pathway. Lysis occurs due to inactivation of the membrane regulators of complement, DAF and CD59, cross-linked by SA. However, monovalent SA attachment does not induce lysis. On the basis of these findings we hypothesized that reduction of the biotin surface density on b-RBC would allow for monovalent SA attachment to b-RBC and that such SA/b-RBC should then be stable in the circulation. In the present work we injected into rats several different radiolabeled RBC probes: rat RBC biotinylated to varying degrees (bn-RBC, where bn represents the input micromolar concentration of biotinylating agent), as well as SA/bn-RBC. Extensively biotinylated rat RBC (b700-RBC, stable in serum in vitro) were rapidly cleared from the bloodstream. We further found that extensively biotinylated human b1000-RBC bound C3b from serum in vitro without detectable lysis, and that rat b700-RBC bound to isolated macrophages in a complement-dependent fashion. Therefore, nonlytic C3b flxation and uptake of C3b-carrying b700-RBC by macrophages appears to be the mechanism leading to clearance of b700-RBC in vivo. Moderately biotinylated RBC (b70-RBC and b240-RBC) were stable in serum in vitro. SA attachment to b240-RBC led to their rapid lysis in serum in vitro, lysis in the bloodstream, and clearance by the liver and spleen. SA attachment to b70-RBC led to fast elimination of SA/b70-RBC from the bloodstream, while in vitro SA/ b70-RBC were stable in serum. Modestly biotinylated RBC (b22-RBC) demonstrated only marginally decreased 60-min survival in the bloodstream regardless of SA attachment. Our in vitro studies indicate that b23-RBC bound approximately 10(5) SA molecules per cell, and the resulting SA/b23-RBC bound 5 x 10(4) molecules of biotinylated IgG (b-IgG) per cell. About 60% of the injected dose of b-IgG/SA/b23-RBC labeled with 51Cr was detected in the rat blood cells 1 day after iv injection. To assess whether b-IgG/SA/b23-RBC circulate in the bloodstream as a stable complex, we have injected 125I-labeled b-IgG/ SA/51Cr-labeled b23-RBC in rats. Up to 60 min after injection, both radiolabels display similar level in bloodstream. Up to 3 h after injection, about 70% of 125I was detected in the blood cells. In contrast, 100% of 125I was detected in plasma after injection of nonconjugated 125I-labeled b-IgG. Thus, major portion of SA/b23-RBC-attached b-IgG circulates as a complex with RBC. About 30% of RBC-bound b-IgG undergoes detachment from the carrier b-RBC, probably in the pulmonary capillaries, because lung level of 125I was twice as high as that of 51Cr. Therefore, the surface density of biotin on the b-RBC membrane appears to play a key role in regulating complement-mediated clearance of bn-RBC and SA/bn-RBC from the bloodstream. Modest biotinylation generates b-IgG/SA/b23-RBC circulating for several hours as stable immunoerythrocytes without detectable lysis or marked elimination, and it may be possible to use these RBC in a drug delivery system.

A biotinylated monoclonal antibody (mAb) to human IgM (b-anti-IgM) has been attached to human red blood cells (RBC) by two different approaches. The first method is performed with biotinylated RBC (b-RBC) and involves stepwise binding of streptavidin (SA) to b-RBC followed by addition and binding of specific b-anti-IgM or b-IgG. b-RBC were prepared with differing input levels of biotin N-hydroxysuccinimide ester (BNHS). At moderate BNHS levels (100 microM) the resulting b-RBC (designated b4-RBC) bound 50,000 molecules of b-IgG after treatment with SA. However, at high BNHS levels (> 1000 microM) the resulting b-RBC bound b-IgG poorly, presumably due to multivalent binding of each SA to several biotins in close proximity on the RBC. b-RBC prepared at high BNHS inputs (but not b4-RBC) were lysed by serum plus SA. Stepwise attachment of b-anti-IgM to SA-coated b4-RBC allows binding of up to 6 x 10(4) molecules of b-anti-IgM/RBC. The second method is based on attachment of b-anti-IgM to RBC via CR1, the primate RBC complement receptor. The SA-biotin system is used to prepare bi-specific mAb complexes (heteropolymers) in which a biotinylated mAb to CR1 is cross-linked with b-anti-IgM via SA. Binding of these heteropolymers to RBC via CR1 is specific and saturable and can facilitate binding of up to 2500 molecules of b-anti-IgM/RBC.(ABSTRACT TRUNCATED AT 250 WORDS)

We have used bispecific, cross-linked monoclonal antibodies (heteropolymers, HP) to facilitate rosette formation between human erythrocytes (EH) and dinitrophenylated sheep erythrocytes (DNP-ES) in the absence of complement. The HP contain monoclonal antibodies (mAbs) specific for both the EH C3b receptor (CR1), and the DNP group, and control experiments with homologous competing non-cross-linked mAbs and naive EH and ES confirm the specificity of the rosetting reaction. These results extend our previous studies, of HP-mediated binding of simple protein antigens to EH CR1, to complex particulate antigens and may eventually allow for the targeting and clearance from the circulation of a variety of pathogens associated with infectious disease.

We used fluorescence microscopy, quantitative FACS analyses, and radioimmunoassays to examine the distribution of complement (C3b)-opsonized antibody/dsDNA immune complexes (IC) bound to normal human erythrocytes (RBCs) via immune adherence (IA). IC were detected with fluorescent anti-human IgG, and the RBC IA-receptor (CR1) was revealed with monoclonal antibodies (Mabs) to CR1 and fluorescent anti-mouse IgG. Under saturating conditions RBCs exhibit a large heterogeneity in binding; a significant fraction binds no IC. The positions of bound IC coincide with CR1 clusters on RBCs, as predicted by several investigators. FACS experiments indicate an excellent correlation between CR1 number and IC binding within an RBC population. The number of CR1 clusters able to bind IC is proportional to the number of CR1 per RBC. However, IC (fluorescent spots) detected per RBC (on average less than 10) are less than the average number of IC bound (20-30). This suggests that each fluorescent spot represents a small number of aggregated IC bound to a CR1 cluster. These "patches" of aggregated complexes may facilitate transfer of RBC-bound IC to cells of the mononuclear phagocytic system. Finally, not all the CR1 clusters bind IC, suggesting that proper geometric alignment of multiple C3b per IC with several CR1 in the cluster is required for IA.

Childhood malaria caused by Plasmodium falciparum (pf) is often characterized by severe anemia at low parasite burdens; the mechanism(s) responsible for this pathology remain to be defined. We have reported that erythrocyte (E) CR1, the immune adherence receptor specific for C3b, is reduced during anemia in childhood malaria, suggesting a possible role for complement in E destruction. Intravascular lysis of infected E by pf leads to release of E breakdown products hemoglobin and hematin, which have inflammatory properties. Free hematin can bind to E, and we find that in serum and in whole blood anti-coagulated with lepirudin, moderate concentrations of hematin activate the alternative pathway of complement and promote deposition of C3 activation and breakdown products on E. We documented C3 deposition by flow cytometry, and additional fluorescence microscopy studies revealed that most of the deposited C3 fragments are located in close juxtaposition to CR1. Western blots confirmed that the C3 fragments are indeed covalently bound to the E, and immunoprecipitation experiments indicated that a fraction of the deposited C3 is covalently bound to CR1. The degree of C3 fragment deposition is directly correlated with E CR1 levels, both within a given donor’s E population and when E from different donors are compared. E opsonized with complement in the presence of hematin form rosettes with Raji cells, through interaction with CR2, the C3dg receptor expressed on several types of B cells including splenic marginal zone B cells. Thus, hematin-mediated complement activation and C3 fragment deposition on E may promote accelerated splenic (or liver) clearance of the youngest E, which have the most CR1, leading to sudden onset of anemia along with reduction of mean CR1 on surviving E. A monoclonal antibody specific for C3b, mAb 3E7, previously demonstrated to inhibit the alternative pathway of complement, completely blocks the C3 fragment deposition reaction. Use of this monoclonal antibody in non-human primate models of malaria may provide insight into mechanisms of erythrocyte destruction and thus aid in the development of therapies based on inhibiting the alternative pathway of complement.

The effects of varying concentrations of heparin and suramin on the complement-mediated binding of antibody/double-stranded DNA immune complexes to red blood cells (RBCs) and Raji cells have been investigated. If the immune complexes are briefly opsonized with complement, suramin can block binding to both cell types, and heparin can block binding to RBCs. In addition, if these complexes are first allowed to bind to RBCs or Raji cells, relatively brief incubations in suramin are sufficient to cause release of the complexes from the cells' C3b receptors. The potential clinical and diagnostic implications of these findings are discussed.

A novel fluorescence method has been developed which should have general applicability for the study of protein-nucleic acid interactions. The association between proteins covalently labeled with dansyl chloride and DNA noncovalently labeled with acridine orange can be studied by monitoring the transfer of fluorescent energy from the donor (the dansyl label) to the acceptor (acridine orange). This method has been illustrated in model fluorescence studies which yield estimates of the combining ratio of histone VIIIS (Sigma, arginine rich) with DNA.

We describe an in vitro assay to study complement fixation of antibody/dsDNA immune complexes formed from SLE sera and radiolabeled dsDNA. The method measures the amount of radiolabeled dsDNA which is part of an immune complex bound to red blood cells via the C3b complement component receptor. The assay is dependent upon active complement, red blood cells, and anti-dsDNA antibodies, but it is independent of the red blood cell donor (type O) and the age of the red blood cells (up to 10 days). The method has been compared in some detail with the Farr assay with respect to the antibody/dsDNA ratio in the immune complexes and the relative stability of the complexes as measured by their resistance to dissociation by excess unlabeled dsDNA. Our results indicate that multiple binding of antibodies to dsDNA is required for complement fixation, and that a significant percentage of those antibodies which fix complement are of high avidity. Finally, a double label assay using both [3H]- and [14C]-dsDNA indicates that the complement fixing potential of the anti-dsDNA antibodies in an SLE serum is strongly influenced by the order of mixing of the isotopes with the serum. The DNA isotope which is added to the serum first is considerably more effective at fixing complement than the isotope which is added 1 h later. The implications of these results with respect to the pathogenesis of SLE are discussed.

The conformational stability of the combining sites of high affinity anti-TNP antibodies in the presence of SDS has been investigated by using several novel fluorescent and spectroscopic probes. Kinetic studies employing these probes along with circular dichroism measurements indicate that the binding of SDS monomer to hydrophobic sites on the antibody molecule causes a very rapid increase in the accessibility of the combining site to water. The loss in the ability of these antibodies to bind hapten in SDS solution appears to depend upon much slower conformational transitions; however, the presence of bound homologous hapten is observed to enhance the resistance of the anti-TNP combining site to denaturation by SDS.

We have examined in mice the immune adherence (IA) reactivity and the fate (clearance kinetics and organ distribution) of a variety of preformed and nascent soluble antibody/125I-DNA immune complexes prepared at antibody excess. Parallel studies of the clearance kinetics and organ deposition of free DNA were also conducted. Preformed immune complexes prepared with large dsDNA bound to mouse platelets in vivo and exhibited clearance kinetics in general agreement with our previous observations in rabbits. Very little kidney deposition was evident for these immune complexes, but three to four times as many counts were found in the kidneys when free DNA was injected. Nascent immune complexes did form in the circulation, but in contradistinction to our previous studies of nascent immune complexes in rabbits and rhesus monkeys, these immune complexes did not demonstrate IA and had clearance kinetics and organ distributions intermediate between the preformed immune complexes and free DNA. Thus, complement-fixing antibody/DNA immune complexes that could form in the circulation might be cleared both via immune complex recognition mechanisms and via DNA recognition mechanisms. The role of IA in modulating the organ deposition of complement-fixing immune complexes is also discussed.

A polyethylene glycol (PEG) radioimmunoprecipitation assay for human IgG is described that is sufficiently sensitive to detect 0.5 ng of IgG. This model antibody-antigen system was also used to study the stoichiometries of PEG-precipitated complexes. Our results suggest that the presence of PEG may affect the stoichiometry of the complexes which precipitate from solution.

The hydration of dioleoyl lecithin (DOL) and dimyristoyl lecithin (DML) has been measured as a function of temperature between −15 and −30 °C, using low-temperature proton magnetic resonance. The hydration of DOL is considerably higher than that of DML. We detect 9 mol of unfrozen water/mol of phospholipid at −25 °C (our “standard” temperature) for DOL, and only 6 mol of water/mol of phospholipid for DML. The gel-to-liquid crystalline phase transition in DOL centered at ca. −19 °C is manifested by a 70% increase in hydration for both vesicles and dispersions. Preparations of either DML vesicles or vesicles of DOL which contain 33 mol% cholesterol would not be expected to undergo this phase transition, and the hydration increase observed for these preparations in the same range of temperature is less than 20%.

The present study evaluated the participation of the primate erythrocyte immune complex (IC) clearing mechanism in the clearance and organ uptake of double-stranded DNA (dsDNA) and of soluble ICs formed with human anti-DNA antibodies and dsDNA (dsDNA-ICs). Five baboons received 51Cr-labeled autologous erythrocytes and after a period of equilibration received separate intraarterial injections of [125I]free dsDNA and [125I]dsDNA-IC. Four of these five baboons were studied on a second occasion. To assess clearance from the arterial circulation and organ uptake, multiple blood samples were obtained from aorta, hepatic vein, and renal vein after injection of each probe. Two minutes after injection, a mean of 85% of dsDNA-ICs were bound to erythrocytes. By contrast, free dsDNA did not bind significantly to blood cells. The clearance rate of dsDNA-ICs from the arterial circulation was significantly faster than that of free dsDNA in all animals but one. Erythrocyte-bound dsDNA-ICs were cleared at a rate similar to that of total dsDNA-ICs. The liver was the major site of uptake of free dsDNA and of dsDNA-ICs. The hepatic uptakes of free dsDNA ( 17 ± 8% 5 min ) and dsDNA-ICs ( 27 ± 8% 5 min ) were not significantly different. 51Cr-labeled erythrocytes were not sequestered in the liver. There was not detectable uptake of free dsDNA or dsDNA-ICs by the kidney but with one exception. Thus, the primate erythrocyte IC clearing mechanism is involved in the clearance of dsDNA-ICs from the circulation but not in the clearance of free dsDNA.

Stopped-flow studies of the refolding of iodoacetamide-blocked bovine serum albumin from the acid unfolded “F” state have been performed. If the protein is incubated with low concentrations of perchlorate anion then the refolding kinetics follow a simple first-order process. The dependence on pH of both the amplitude of the observed transients and the measured rate constants indicates that the N ⇌ F transition is highly cooperative. The results are consistent with the postulated multidomain structure of albumin which has been developed as a result of both sequence work and a variety of physical studies.

The reaction of N-dansylaziridine with serum albumin (both bovine and human) results in incorporation of about 3 mol of covalently bound dansyl label per mol protein. This indicates that a number of nucleophilic groups in these proteins (in addition to the free sulfhydryl group) will react with this reagent. The reaction has been studied in detail for bovine serum albumin and the results suggest that one of the sites labelled by the reagent may be at the unusual “catalytic site” responsible for the enzyme-like activity of bovine serum albumin recently described (Taylor, R.P., Chau, V., Bryner C. and Berga S. (1975) J. Am. Chem. Soc. 97, 1934–1942). The reaction of N-dansylaziridine with a variety of other proteins indicates a pattern of labelling consistent with high specificity for the sulfhydryl group. The explanation for the unexpected excess reactivity of albumin with the “sulfhydryl specific” reagent N-dansylaziridine must be related to the three-dimensional structure in albumin which enables a number of specific residues to manifest unusually high degrees of nucleophilic reactivity.

Water which remains unfrozen at −25 °C in the presence of phosphatidyl choline (PC) gives rise to a proton magnetic resonance signal which can be used to measure the hydration of single-walled vesicles and multilamellar liposomes of PC. The proton magnetic resonance signal of the unfrozen water in these systems is strongly dependent upon the nature of the molecular domain in which the water is situated. For example, at cholesterol to PC molar ratios below 35 mol%, the vesicle hydration signal consists of a relatively narrow symmetric peak (line width, ~150 Hz). At higher molar ratios, however, rather broad asymmetric signals appear (line widths, ~300–1000 Hz) which indicate that when significant quantities of cholesterol are packed in the bilayer there must be regions in which there is a preferred direction for motion of the unfrozen water. It is possible to solubilize significant quantities of cholesterol by sonicating it in concentrated solutions of sodium dodecyl sulfate. Addition of cholesterol to PC vesicles via these sodium dodecyl sulfate-cholesterol complexes caused hydration changes in the PC, which, at high cholesterol to PC molar ratios, paralleled the effects of cholesterol on PC hydration in homogeneous vesicles in which the cholesterol and PC were simply cosonicated.

We have prepared and characterized four monoclonal antibodies (MAbs) to human C3b of high specificity and affinity. Our procedure did not require a purified source of C3b for immunization. Instead, C3b and C3bi were deposited on Sepharose 4B via the alternative pathway of complement activation in normal human serum, and this C3b(i)-Sepharose served as the immunogen. C3b(i)-Sepharose was also prepared from a number of primate and non-primate sources, and this allowed us to demonstrate that the anti-human C3b MAbs cross-reacted with primate-derived C3b, but not with C3b from non-primates. The procedures we have developed may be useful in the further investigation of species-specific C3 fragment-binding proteins from both primate and non-primate sources.