Paroxysmal nocturnal haemoglobinuria treatment with eculizumab is associated with a positive direct antiglobulin test.

BACKGROUND/OBJECTIVES: Paroxysmal nocturnal haemoglobinuria (PNH) is characterized by intravascular haemolysis with a negative direct antiglobulin test (DAT). Eculizumab is a humanized monoclonal antibody that inhibits complement component C5 and is approved for PNH treatment. Recent publications demonstrated that some patients with PNH develop a positive DAT during eculizumab treatment. These published clinical trials investigated a highly selected patient population. Therefore, it seems important to study this topic in a general PNH patient population with a longer follow-up. MATERIALS AND METHODS: We analysed haemolytic activity, RBC transfusion requirement, effect on DAT and ferritin levels in 41 patients with PNH before and during eculizumab therapy with a median follow-up of 24 months (range 1-63 months). RESULTS: During eculizumab therapy, median LDH decreased (1657-258 U/l; P < 0·0001), while median haemoglobin increased (9·2-10·3 g/dl). Eighteen of 32 pts (56%) who previously required regular transfusions became transfusion independent. DAT was positive for C3d in 72·4% of 21 eculizumab-treated pts with available DAT. Ferritin levels increased (69-348 ng/ml, P < 0·0001). This increase was more pronounced in pts with ongoing transfusion dependency during eculizumab therapy. CONCLUSION: Eculizumab therapy for PNH should be added to the list of possible causes for a positive DAT. Intravascular haemolysis was inhibited by eculizumab, but signs of extravascular haemolysis should be monitored. Because renal iron loss was stopped, eculizumab-treated pts can be prone to iron overload and therefore ferritin concentrations should be monitored closely.

Release of mediators of systemic inflammatory response syndrome in the course of a severe delayed hemolytic transfusion reaction caused by anti-D.

BACKGROUND: In vitro studies suggest that mediators of systemic inflammatory response syndrome are generated in the course of hemolytic transfusion reactions. Evidence for the in vivo significance of these findings is given by the present clinical and laboratory analysis of a severe delayed hemolytic transfusion reaction (DHTR). CASE REPORT: A 67-year-old patient (blood group O, D-negative) with a negative pretransfusion antibody screen received a massive transfusion because of arterial bleeding (Day 1). The transfusion of group O, D-positive red cell concentrates was unavoidable because of limited supplies. At Day 10, the patient developed a DHTR with symptoms of septic-toxic syndrome and signs of hemolysis; he received an exchange transfusion. Serologic markers, as well as proinflammatory and anti-inflammatory mediators, were monitored at the onset of the DHTR and during the exchange transfusion. RESULTS: At Day 10, the direct antiglobulin test was positive; anti-D was present, most likely as the result of an anamnestic immune response. Interleukin (IL)-1 was not detectable; all other mediators monitored were elevated: IL-1 receptor antagonist, tumor necrosis factor, IL-6, IL-8, IL-10, neopterin, elastase, C3a-desArg, C-reactive protein, and fibrinogen. Most of the values declined during the exchange transfusion, which was followed by an improvement of the clinical presentation. CONCLUSIONS: Mediators of systemic inflammatory response syndrome were released in the course of a DHTR caused by anti-D. Severe clinical symptoms could be treated successfully by exchange transfusion.

Immunocompetence of T lymphocytes during whole-blood storage.

In WB stored for 28 days at 4 degrees C the pattern of peripheral T-cell CD25 (IL-2R) expression after polyclonal in vitro stimulation was examined. Flow cytrometic analysis was performed with lymphocytes after 72 h in culture with and without ConA using antibody against CD3 and CD25. In culture supernatants IL-2 was determined. T-cells bearing the CD25 marker decreased with storage time, the mean fluorescence (i.e., number of IL-2R molecules per cell) remained rather stable. IL-2 concentrations peaked with stimulated cells which have been stored for 6 days. The low detectable levels of IL-2 in cultures of T-cells which have not been stored may reflect a much more efficient interplay of IL-2/IL-2R. Thus, even after longer periods of blood storage there are sufficient numbers of T-cells which can be stimulated to excite preprogrammed effector functions, i.e. also a GVHD.

Non-enzymic activation of the fifth component of human complement, by oxygen radicals. Some properties of the activation product, C5b-like C5.

Purified human C5 was converted non-enzymically to an activated form as defined by its ability to participate in reactive lysis. This conversion occurred following exposure to systems that generate oxygen radicals, namely addition of H2O2 in the presence of ascorbic acid and iron or the addition of xanthine oxidase, acetaldehyde and iron. The conversion of C5 to a functionally active species was iron-dependent and inhibited by hydroxyl radical scavengers such as DMSO. The findings suggest that OH. is the active oxygen species that converts C5. The conversion product of C5, termed C5(H2O2), is C5b-like due to its ability to bind C6 and cause reactive lysis. C5(H2O2) is much more stable than C5b obtained by complement convertases. Although C5(H2O2) has lost the binding site of native C5 for C3b it can be cleaved by complement-derived convertases; the cleavage is, however, less efficient than in the case of native C5. The resulting cleavage product, which is C5a-like, is chemotactic although C5(H2O2) is not chemotactic. C5(H2O2) serves as a better substrate for plasma kallikrein than native C5, resulting in the generation of a C5a-like chemotactic product. These data indicate that oxygen radicals can bring about a conformational change in C5, causing it to behave as a functionally activated molecule of the complement system. This may have implications for the role of complement and its activation in the inflammatory response.

Activation of the alternative pathway of human complement by sulfhydryl compounds of analytic and therapeutic use.

Thiol-containing drugs (dimercaprol, dimercaptopropanesulfonate, captopril, penicillamine, N-acetylcysteine) and the standard reducing agent beta-mercaptoethanol, activate the alternative pathway of complement as shown by in vitro experiments. Depending on the substance tested, at concentrations of 0.5-5 mM, cleavage products of C3 and factor B were demonstrable in serum by immunoelectrophoresis. The regulatory protein factor I proved to be very sensitive to thiols; this observation offers an explanation for the alternative pathway activating effect of these substances. At concentrations of thiols that initiate the alternative pathway, the classical pathway was not or only to a minor extent activated; however, the activity of C2, C5 and one or several of the components C6-9 was directly affected. Alkylation of the thiol group of the compounds tested, abrogated their effects on the complement system.

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.

Generation of anaphylatoxin activity related to C3a, by treatment of human serum with the nitrogen nucleophile N2H4 or the chaotrope KSCN.

The present study is concerned with the proteolytic processing of complement component C3 in normal human serum treated with N2H4 or KSCN in the presence of EDTA. Upon incubation with these agents, C3 is first converted to the thiolester-cleaved form (C3i) and thereafter fragmented by factor I. In consecutive, relatively slow steps, spasmogenic and platelet-aggregating activity is released. The active principle shows characteristics of C3a. First, the pretreated sera deactivate guinea pig ileum and platelets towards the action of C3ahog, but not C5a-desArghog. Second, the activity is only stable under conditions causing inhibition of serum carboxypeptidase N, such as in the presence of EDTA or of MERGETPA (DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid). Third, the molecular weight determined by gel filtration is in agreement with that of C3a. The release of C3a activity requires conversion of C3 to C3i, as well as the complement-independent generation of proteolytic activity in the pretreated sera. The enzyme releasing C3a activity is a serine esterase probably identical with Hageman factor, kallikrein, or another protease related to the contact system.

Mechanisms of complement activation by crystalline cholesterol.

The mechanism by which cholesterol crystals activate complement in human serum has been studied. Crystals treated with serum and washed with buffer contain a fixed C3/C5 convertase. Its generation is dependent on the presence of divalent cations (and of factor B). The cholesterol-fixed convertase is subject to decay and can be regenerated by factors B and D. C2 in combination with C1 is not essential but enhances the convertase formation. These findings indicate that it is predominantly the alternative C3/C5 convertase C3bBb(P) that assembles on cholesterol during exposure to human serum. By the use of different antisera and immunofluorescence a C3 fragment, probably C3b, was demonstrated on serum-treated crystals. Its fixation is resistant to washing with urea, and with buffers of differing pH: by hydroxylaminolysis the C3 fragment dissociates from the crystals. This indicates a covalent ester bond linking the labile binding site of activated C3 to the hydroxyl group of cholesterol. Cholesterol acetate does not fix C3 nor acquire a C3-cleaving activity upon contact with serum. In addition, cholesterol crystals bind factor I (C3b inactivator) and in this way may facilitate fixation and amplification of the alternative C3/C5 convertase.

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.

Effect of metrizamide, a nonionic radiographic contrast agent, on human serum complement. Comparison with ionic contrast media.

The nonionic radiographic contrast material (RCM) metrizamide causes consumption of total complement activity in normal human serum (NHS) in vitro in the absence and to a lesser extent also in the presence of EDTA. The depression of titers of total complement is related to an inactivating effect of metrizamide on component C2. Furthermore, metrizamide induces activation of the alternative pathway as evidenced by the appearance of C3 and factor B cleavage products in NHS, dependent on the presence of divalent cations. Alternative pathway activation is probably mediated by an antagonizing effect of metrizamide on the inactivation of C3b. Unlike ionic RCM, the nonionic substance metrizamide does not lead to cleavage of the internal thiolester bond present in native C3 and C4, at concentrations that produce potent consumption of C3 activity in NHS.

Effect of different penicillin derivatives on complement components in human serum.

The effect of different penicillin derivatives on the human complement system has been studied in vitro. Four penicillins tested (benzylpenicillin, oxacillin, methicillin, azlocillin) were found to inactivate total hemolytic complement dose dependently. This effect is caused by direct inactivation of C2, C5 and one or more of the components C6-9 as well as by activation of the alternative pathway. At relatively high concentrations, penicillins induce a conformational change in C3 and C4, leading to the C3b-like or C4b-like forms of these molecules, respectively. Prominent effects on the complement system occur at penicillin concentrations exceeding those achieved in plasma under regular therapeutic use. However, a synergism of penicillins with drugs acting similarly on the complement system, such as iodinated radiographic contrast media, may be expected.

Effect of radiographic contrast media on complement components C3 and C4: generation of C3b-like C3 and C4b-like C4.

Various water soluble iodinated radiographic contrast media (RCM) have been studied for their effect on complement components C3 and C4, purified and in serum. Hepatotropic RCM, and at higher concentration also some nephrotropic RCM, were found to exert a direct effect on purified C3 and C4. RCM treated human C3 and C4 are characterized by (a) loss of haemolytic function, (b) retention of activity in the formation of fluid phase C3 convertases and (c) an antigenic relationship to activated C3 and C4 (C3b and C4b, respectively). This direct alteration of C3 and C4 can probably also occur in serum since loss of haemolytic function is observed at similar RCM concentrations after incubation of serum and of purified components. It is concluded that RCM treated C3 and C4 represent altered forms of these components that resemble C4b and C3b in activity and conformation (C3b-like C3 and C4b-like C4). The alteration is probably caused by binding of RCM, exerting a mild denaturing effect. C3b-like C3 is a potential activator of the alternative pathway, and both C3b-like C3 and C4b-like C4 are known to be cleaved by serum inactivators. A possible pathological significance of the generation of C3b-like C3, C4b-like C4 and their split products remains to be evaluated.

Chain structure of cobra venom factor from Naja naja and Naja haje venom.

The chain structure of cobra venom factor, whether isolated from Naja naja venom (CVFn) or from Naja Haje (CVFh) is similar. Both homologous proteins are composed of three disulphide-linked chains (A, B, and C) with apparent molecular weights of 72,000, 54,000, and 27,000-35,000 for CVFn and 68,000, 51,000 and 30,000-32,000 CVFh. That all three polypeptides are integral parts of CVF was demonstrated by investigation of the chain pattern after partial reduction. Reduction with 1-2 mM dithiothreitol under non-denaturing conditions yielded free B-chain, together with an intermediate product composed of disulphide-linked A- and C-chains. The C-chain was heterogenous when investigated by electrophoresis in polyacrylamide slab gels in the presence of SDS. Similarly, isoelectric focusing of CVFn and CVFh showed a multiplicity of bands in the pH range 5.2-6.4. Limited tryptic digestion resulted primarily in the fragmentation of the B-chain. CVFh is much more sensitive to tryptic attack than CVFn. In all our preparations of CVFh a partial, trypsin-like fragmentation of the B-chain was detectable to various extents.

The fourth component of human complement treated with amines or chaotropes or frozen-thawed (C4b-like C4): interaction with C4 binding protein and cleavage by C3b/C4b inactivator.

We have shown previously that C4 treated with amines or chaotropes, although uncleaved, exhibits properties that are similar to C4b. Studies by other groups suggest that this C4b-like form of C4 is characterized by the lack of an internal thiolester bond that is present in native C4. We report here that C4 treated with N2H4 or KSCN or frozen-thawed, unlike native C4, forms a complex with C4-binding protein (C4-bp) and is cleaved by C3b/C4b inactivator (I). Fragmentation of C4b-like C4 by I occurs without previous cleavage to C4b and requires the presence of C4-bp. Cleavage of C4b-like C4 proceeds in two steps: a small fragment (16,000 m.w.) is released first, followed by cleavage of the remaining alpha-chain fragment (83,000 m.w.) into polypeptides of 46,000 (C4d) and 32,000 m.w. All fragments, except the 46,000 m.w. fragment, are disulfide-linked to the beta- and/or gamma-chains of C4. Cleavage of C4b-like C4 probably occurs at the same points in the alp a-chain as in C4b; however, C4b-like c4 also contains C4a. Based on the m.w. determinations, the C4a portion of the alpha-chain is present in the 83,000 m.w. fragment, and after the second cleavage, in the 32,000 m.w. fragment of C4b-like C4. These findings suggest the following alignment of alpha-chain fragments: the N-terminal C4a portion is attached to a polypeptide of about 25,000 m.w. (alpha 3), which is followed in the sequence by C4d (alpha 2) (46,000 m.w.) and a small 16,000 m.w. fragment (alpha 4) that forms the C-terminus.

Conformational changes in complement component C4 induced by activation, treatment with amines, chaotropes, or freezing-thawing, detectable by radioiodination using lactoperoxidase.

Conversion of C4 to C4b by enzymic cleavage or generation of a C4b-like form by treatment with amines, chaotropes, or freezing and thawing is accompanied by conformational alterations in the molecule. The nature of the changes is the same whether C4 is converted to a C4b-like form or to C4b. Evidence for structural changes at the surface of the C4 molecule was obtained by peripheral labeling with 125I by the lactoperoxidase method: there are residues, probably tyrosines, that are inaccessible for radioiodination in C4b and C4b-like C4, but exposed in native C4. The residues concerned are located in two separated parts of the alpha-chain at a distance of approximately 9000-34,000 m.w. from the N-terminus and in a 16,000 m.w. portion at the C-terminus. It is so far unknown whether the observed changes are involved in the uncovering of new binding sites, or whether they are the mere reflection of alterations elsewhere in the molecule that are of greater functional significance.

Isolation and properties of a complement inhibitor from Naja haje venom, distinct from known anticomplementary factors in cobra venom.

A complement inhibitor (CI) has been isolated from cobra (Naja haje) venom which is distinct from the two known anticomplementary factors in cobra venom [1], in functional properties as well as structure. CI is a small (mol. wt 26,000, determined by sodium dodecyl sulphate gel electrophoresis), heat-labile glycoprotein; the amino acid composition is that of a globular protein. CI interferes at various steps of the complement sequence, including reactions of the classical and alternative pathway. No effect was observed on C4 fixation and on the assembly of the membrane attack complex from C6-9 (minor inhibiting effects, if present, have not been excluded). Initiation of the alternative pathway is inhibited by CI already at the stage of cleavage of factor B. CI binds to C4, C4b, C3 and C3b; since the major inhibitory action of CI is lost after washing of cell intermediates, complex formation and, as a consequence, steric hindrance may be responsible for the inhibiting effects of CI. CI also interferes with binding of C3b to C3b receptors on human erythrocytes. CI is non-toxic in mice when given intraperitoneally in doses of 5 microgram/g.

Treatment of human complement components C4 and C3 with amines or chaotropic ions. Evidence of a functional and structural change that provides uncleaved C4 and C3 with properties of their soluble activated froms, C4b and C3b.

Treatment of human components C4 and C3 with amines like hydrazine, ammonium hydroxide, and neutral ammonium salts or with chaotropic salts like KSCN and NaBr leads to complete loss of haemolytic activity. The pretreated components are, however, still active in formation of soluble C3 convertases. This activity pattern is reminiscent of the activities of C4 and C3 that have been activated by cleavage in the fluid phase. Indeed, the antigenic properties of pretreated C4 and C3 are similar to soluble C4b and C3b. The polypeptide chain structure of pretreated C4 and C3, is, however, identical to that of the untreated components when investigated by SDS gel electrophoresis. Pretreatment even reduces greatly the susceptibility of C4 to cleavage by C1s and of C3 to cleavage by classical and alternative pathway C3 convertases. Pretreated components have lost the ability to combine with EAC1 and EAC142, respectively; this fact explains their failure to exhibit haemolytic activity. In serum, pretreated C4 and C3 are cleaved in a manner similar to C4b and C3b. Amines and chaotropic ions cause the same functional and structural alterations, which are best explained by assumption of a conformational change. A similar transformation can also occur in C4 and C3 during preparation or storage.