Complement Split Product Research Articles

Inhibition of complement activation: a novel mechanism for the protective effects of heparin in antiphospholipid antibody-induced pregnancy loss

The antiphospholipid syndrome (APS), defined by thrombosis and recurrent pregnancy loss in the presence of antiphospholipid (aPL) antibodies, is generally treated with anticoagulation. Because complement activation is essential and causative in aPL antibody-induced fetal injury, we hypothesized that heparin protects mice from fetal loss in APS by preventing complement activation on trophoblasts and that anticoagulation, per se, is not sufficient to prevent miscarriage. We used a murine model of APS in which pregnant mice are injected with human IgG-containing aPL antibodies (aPL-IgG) or IgG from healthy individuals (NH-IgG). Passive transfer of aPL-IgG caused a 43.3 ± 3.5% frequency of fetal resorption (P < 0.001 versus NH-IgG) and treatment with either unfractionated heparin (UFH) (10 U or 20 U subcutaneously twice daily) or low molecular weight heparin (LMWH) (enoxaparin) reduced the frequency of fetal resorption to that of mice treated with NH-IgG: UFH10 + aPL, 9.6 ± 3.2; UFH20 + aPL 10.4 ± 2.1; LMWH + aPL, 11.8 ± 2.8; NH-IgG, 10.5 ± 2.5% (P < 0.01 versus aPL). Even in the absence of detectable anticoagulation, as in mice treated with 10 U UFH, heparins prevented aPL-induced pregnancy loss and inhibited systemic complement activation evidenced by generation of C3adesArg (aPL, 1212 ± 101* ng/ml; UFH10U + aPL, 178 ± 25; LMWH + aPL, 180 ± 15; NH-IgG, 215 ± 381, * P < 0.05 versus NH-IgG). Heparin limited C3 deposition in deciduas of mice treated with aPL. Neither fondaparinux nor hirudin inhibited the generation of complement split products or prevented pregnancy loss, despite anticoagulation levels comparable with heparin, indicating that anticoagulation is insufficient therapy for APS-associated miscarriage. We assessed the effects of anticoagulants on complement activation in vitro using BeWo cells, trophoblast-like cells with externalized phosphatidylserine recognized by aPL. APL-opsonized BeWo cells cultured with mouse serum activated complement, evidenced by increased surface-bound C3 fragments (detected as C3 staining by FACS) and generation of soluble C3adesArg in supernatants. In the presence of UFH or LMWH, however, complement activation was completely inhibited (C3adesArg [ng/ml]: aPL, 680 ± 30; UFH + aPL, 120 ± 20*; LMWH + aPL, 105 ± 18*; % C3-positive BeWo cells by FACS: aPL, 31 ± 11; aPL + UFH, 9 ± 5*; LMWH + aPL, 5 ± 3; * P < 0.01 versus aPL). In contrast, neither fondaparinux nor hirudin inhibited complement activation in vitro. In conclusion, heparins inhibit complement activation in vitro and in vivo. Heparins may prevent obstetrical complications in APS by blocking activation of complement induced by aPL targeted to deciduas – not by preventing placental thrombosis. These findings explain how subanticoagulant doses of heparin can limit antibody-mediated tissue injury.

C4d: a pathogenetically relevant marker in kidney transplantation

Antibody-mediated allograft rejection should be discriminated from cell-mediated rejection processes since these two distinct pathogenic mechanisms are likely to require different therapeutic approaches. The complement split product C4 d (although biologically inactive by itself) turned out to be a reliable diagnostic marker of antibody-induced complement activation in kidney allografts. In contrast to other organs like heart and bowel, there is no indication that ischemia/reperfusion injury might induce the classical pathway of complement activation in the kidney. Endothelial C4 d deposits in peritubular capillaries of transplanted kidneys are therefore robust indicators of antibody-mediated alloreactivity with potentially unfavourable outcome. The high efficiency of therapeutic strategies aimed at removing alloantibodies from the recipient's circulation (i.e. immunoadsorption and plasmapheresis) in cases of C4d positive rejection further underscores the important pathogenic role of antibodies in renal allograft rejection and the diagnostic relevance of endothelial C4 d deposits. C4 d deposits can also occur de novo in late allograft biopsies. Their association with basement membrane injury in peritubular capillaries and glomeruli, which are the hallmarks of chronic rejection, indicates a contribution of humoral immune reactions to chronic rejection. Due to the clinical relevance of humoral rejection and due to the fact that the latest update of the Banff classification introduced the pathogenically based subdivision of acute rejection into a cell-mediated and an antibody-mediated variant every renal allograft biopsy should be stained for C4 d. It is important to be aware that humoral and cellular rejection often occur simultaneously and that C4d deposits alone are not an unequivocal proof of humoral rejection that immediately requires treatment.

Diagnosis and treatment of antibody-mediated kidney allograft rejection.

Evidence of a significant pathogenetic role of donor-reactive antibodies (DRA) in kidney allograft rejection is accumulating. At least, partially owing to the recent discovery of the complement split product C4d as a valuable rejection marker, antibody-mediated rejection (AMR) has regained increasing attention. We review here the value of various diagnostic criteria, including immunohistochemistry (C4d staining), histomorphology and posttransplant serology, for the diagnosis of AMR. Furthermore, the mechanisms underlying alloantibody/complement-mediated allograft injury are discussed in detail. Finally, a thorough discussion of recently proposed "anti-humoral" therapeutic strategies is provided.

C4d complement split product expression in chronic rejection of renal allograft

Chronic allograft rejection remains the major cause of late renal graft loss. Its pathogenesis is complex, depending on both immunological and nonimmunological factors. An important role in development of chronic rejection is ascribed to an ongoing immunological reaction mainly of the humoral type. C4d complement split product, as a stable fragment of complement degradation activated by antigen-antibody complexes, is considered to be an indicator of humoral activity in allografts. The aim of the present study was to establish a correlation between C4d expression and morphological findings specific for chronic rejection among biopsy specimens from patients with deteriorating graft function versus protocol biopsy specimens versus biopsy specimens of native kidneys with glomerular diseases. C4d deposits in peritubular capillaries and glomeruli were observed in 83% of patients with morphological changes of chronic rejection. No C4d expression was found in the protocol biopsy group. C4d deposits in glomeruli localizations were found in kidneys from patients with glomerulopathies; the pattern of distribution was similar to that for antibodies characteristic for glomerulonephritis. There was a positive correlation between C4d expression and morphological features of chronic rejection. In our opinion, only peritubular capillary localization is specific for a rejection process; glomerular localization is nonspecific and probably secondary to antigen-antibody complex deposition in course of some types of glomerulopathies.

Complement activation in a model of chronic fatigue syndrome

Background: A need exists to identify biological markers in chronic fatigue syndrome (CFS). Objective: To use an exercise and/or allergen challenge to induce the symptoms of CFS and to identify a biological marker that correlates with these symptoms. Methods: Patients with CFS (n = 32) and age-matched, normal control patients (n = 29) exercised for 20 minutes on a stationary bike at 70% of their predicted max work load (Watts). Patients from each group with positive skin test results were also challenged with intranasally administered relevant allergens. Symptoms were recorded for 2 weeks before and 1 week after each challenge, using 3 different instruments. Blood samples were taken before, and 0, 1, 6, and 24 hours after challenges. Levels of complement split products, cell-associated cytokines, and eosinophilic cationic protein were measured. Mean preexercise and postexercise symptom scores were evaluated for each group. Results: Exercise challenge induced significant increases of the complement split product C4a, but not C3a or C5a, at 6 hours after exercise only in the CFS group ( P < .01), regardless of allergy status. Mean symptom scores were significantly increased after exercise through the use of a daily diary ( P < .03) and a weekly diary ( P < .01) for the CFS group only. Mean scores for the Multidimensional Fatigue Inventory categories “reduced activity” and “mental fatigue” were significantly increased in the CFS group only ( P < .04 and P < .02, respectively). Conclusions: Exercise challenge may be a valuable tool in the development of diagnostic criteria and tests for CFS. Establishment of a role for complement activation products as markers or participants in production of illness require further study. (J Allergy Clin Immunol 2003;112:397-403.)

Inflammatory mediators in autotransfusion drain blood after knee arthroplasty, with and without leucocyte reduction.

The aim of this study was to evaluate whether leucocyte-reducing filters influenced complement activation and the formation of pro-inflammatory cytokines in autotransfusion drain blood after knee arthroplasty. Twenty-three patients undergoing knee arthroplasty were divided into two groups. All patients were given salvage blood postoperatively. In Group A, a leucocyte filter was connected between the wound and the drain blood container. In Group B the drain blood was not leucocyte filtered. Complement split products and cytokines were analysed in circulating blood and in drain blood, together with blood-cellular differential counts. Drain blood showed activation vs. venous blood, with elevated concentrations of C3a, SC5b-9, interleukin (IL)-6, IL-8, polymorphonuclear (PMN) elastase and tumour necrosis factor-alpha (TNF-alpha) (P<0.05 to P<0.001). The leucocyte filter reduced TNF-alpha (P<0.01), but triggered complement activation (P<0.05). Room-temperature incubation increased the concentration of IL-8 (P<0.01), which was seen in both venous and drain blood. The leucocyte filter prevented formation of IL-8 (P<0.01). In drain blood at 24 h the inflammatory reactions accelerated (P<0.05-0.001), although the filter reduced the leucocyte counts and TNF-alpha concentrations. The leucocyte filter reduced IL-8 and TNF-alpha in drain blood, but at the same time triggered complement activation. Incubation affected the inflammatory spectrum of both drain blood and control venous blood, and the filtering reduced this activation.

Biologic Limits of Viability

After completing this article, readers should be able to: 1. Define the gestational range for extremely preterm birth. 2. Explain the difference between possibility and probability in reference to survival following extremely preterm birth. 3. Describe the key lung development required for adequacy of gas exchange. 4. Describe the renal functional limitations for water, sodium, and creatinine homeostasis in the first postnatal week of the extremely preterm infant. 5. List at least one structural and/or functional limitation of at least five organ systems that increases mortality risks for the extremely preterm infant. With the advent of neonatal intensive care in the 1960s, preterm infants born at progressively lower gestational ages began to survive. The progression of the decrement did not follow a recognizable relationship; rather, it was erratic as the knowledge base of neonatal physiology and pathology expanded and new technical capabilities for life support were developed. The most dramatic increase in survival of extremely preterm birth (<26 completed weeks’ gestation) came with the introduction of surfactant replacement therapy in the late 1980s. The increased use of maternal antenatal corticosteroid therapy for induction of lung maturation with threatened preterm delivery following the National Institute of Child Health and Human Development Consensus Conference recommendations in 1994 also contributed to improved survival. Since that time, the survival of infants of gestational ages as low as 22 weeks’ has been reported. The purpose of this article is to review the current understanding of the biologic factors limiting survival potential, particularly as they inform clinical decision-making. Viability or survival potential, when used in reference to a fetus, frequently is used to indicate the possibility for a fetus to be live born and capable of living to a specified endpoint of either a designated time, reaching a certain age or a landmark event, admission to a neonatal intensive care unit, …

Non-Invasive Monitoring of Renal Transplant Recipients: Urinary Excretion of Soluble Adhesion Molecules and of the Complement-Split Product C4d

Background: The number of inducible adhesion molecules known to be involved in cell-mediated allograft rejection is still increasing. In addition, recent data describe complement activation during acute humoral allograft rejection. The aim of this study was to assess whether specific molecules from either pathway are excreted into urine and whether they can provide useful diagnostic tools for the monitoring of renal transplant recipients. Methods: Urinary concentrations of soluble adhesion molecules (sICAM-1, sVCAM-1) and of the complement degradation product C4d were determined by standardized ELISA technique in 75 recipients of renal allografts and 29 healthy controls. Patient samples were assigned to four categories according to clinical criteria: group 1: acute steroid-sensitive rejection (ASSR, n = 14), group 2: acute steroid-resistant rejection (ASRR, n = 12), group 3: chronic allograft dysfunction (CAD, n = 20) and group 4: stable graft function (SGF, n = 29). Results: All patients with rejection episodes (groups 1–3) had significantly higher values of urinary sC4d compared with healthy controls and patients with stable graft function (p < 0.05). The urinary levels of sVCAM-1 were significantly higher in group 2 (ASRR) compared with all other groups (p < 0.001). Uniformly low amounts of s-VCAM-1 and complement-split product C4d were excreted by healthy controls (group 0). In contrast, urinary sICAM-1 concentration in healthy controls was almost as high as in group 2 (ASRR) whereas patients with a stable functioning graft (group 4) excreted significantly less sICAM-1 (p < 0.05). Conclusion: The evaluation of sVCAM-1 and sC4d excretion in urine can provide a valuable tool with regard to the severity and type of allograft rejection. With respect to long-term allograft survival, serial measurements of these markers should have the potential to detect rejection episodes and prompt immediate treatment.

Activation of the complement system by different autologous transfusion devices: an in vitro study.

The aim of the present investigation was to study whether autologous transfusion devices activate the complement system and whether complement-activated blood is more vulnerable to further activation during processing. Forty-eight blood units were randomized to be processed by one of three different salvage systems: Group 1 underwent whole blood filtration (hemofiltration) (n=16); Group 2 underwent continuous processing, saline washing, and centrifugation (CATS, Fresenius AG ) (n=16); and Group 3 underwent saline washing and centrifugation (Cell-Saver, Haemonetics Corp.) (n=16). Eight blood units for each system were activated with cobra venom factor (CVF) at a concentration of 0.2 U per mL whole blood before processing. C activation was studied by determinations of C4d, Bb, C3a, and SC5b-9. Samples were drawn from whole blood, processed blood, and the waste bags. The concentrations of Bb, C3a, and SC5b-9 in whole blood after activation with CVF were significantly elevated compared to blood that was not activated (p < 0.01). Processed blood from hemofiltration contained significantly higher levels of complement-split products than techniques that use washing and centrifugation. The concentrations of SC5b-9 in blood processed by hemofiltration were higher in the experiments with CVF activation (p < 0.05). The tested autologous transfusion systems did not themselves activate the complement system, and complement-activated blood was not more vulnerable to further activation during processing. A blood-salvaging technique that used washing and centrifugation reduced elevated concentrations of complement-split products, whereas hemofiltration did not.

Complement in Ischemia Reperfusion Injury

In the current issue of The American Journal of Pathology Stahl et al describe an important role for the alternative complement pathway in ischemia reperfusion (I/R) of the intestine, using factor D-deficient mice. 1 The requirement for the complement system in I/R-induced tissue injury has been established, but the contribution of the various complement pathways (classical, alternative, and lectin), is largely unknown because, until recently, the tools for such an investigation were not available. The complement split product, C5a, and the terminal membrane attack complex, C5b-9 (MAC), are believed to be responsible for complement-mediated I/R-induced tissue injury, and, therefore, may be targets for therapeutic interventions in clinical settings. More precise understanding of the relevant complement pathway(s) involved in the final generation of C5a and MAC will define the options for therapeutic intervention.

Complement activation is responsible for acute toxicities in rhesus monkeys treated with a phosphorothioate oligodeoxynucleotide

The objective of this study was to define the role of complement activation in the acute and transient toxicities associated with administration of phosphorothioate oligonucleotides in monkeys. In the absence of complement inhibitor, complement activation blocker-2 (CAB-2), i.v. infusion of 20 mg/kg ISIS 2302 produced increases in the concentrations of the complement split products Bb and C5a (100- and 7-fold, respectively). Monkeys also experienced marked changes in blood pressure (hypertension and hypotension), clinical signs of toxicity (lethargy and periorbital edema), fluctuations in circulating neutrophil counts, and elevations in serum cytokine levels (45-, 12-, and 4-fold increases in IL-6, MCP-1, and IL-12, respectively). Changes occurred at or near the end of infusion and returned to normal over time. One of the three animals died approximately 4 h following infusion of 20 mg/kg ISIS 2302 alone. In contrast, prior treatment with CAB-2 effectively blocked complement activation, as well as the ISIS 2302-induced hemodynamic and clinical responses. Importantly, plasma concentration of ISIS 2302 were unaffected by CAB-2 pretreatment. Thus, the protection afforded by CAB-2 was due to its inhibition of complement activation rather than to any impact on the disposition of ISIS 2302. These results clearly demonstrate the causal relationship between activation of the alternative complement pathway and the hemodynamic and clinical responses associated with rapid infusion of phosphorothioate oligonucleotides. Demonstration of this relationship underscores the importance of avoiding complement activation in patients to ensure the continued safe use of phosphorothioate oligodeoxynucleotides.

Serum C4 levels in patients with systemic lupus erythematosus in remission

Twenty-six patients with systemic lupus erythematosus (SLE) showing systemic lupus activity measure (SLAM) and SLE disease activity index (SLEDAI) scores ≤2, as well as a lower C4 concentration than the mean C4 levels of healthy controls, were selected to evaluate the C4 levels of SLE patients in remission. Serum complement (CH50), complement components (C4, C3, and B), complement split products (C4d, iC3b, and Bb), phenotypic expression of C4 allotype, C4 production by peripheral blood monocytes, peripheral blood lymphocyte subpopulation, and interferon-gamma (IFN-γ) production were examined. In patients with SLE in remission, the C4 consumption (C4d/C4) was found to increase, and this was considered to be the most important factor for determining the serum concentration of C4. However, the relevance of the C4 allotypic expression was minimal. The IFN-γ-stimulated production of C4 by peripheral blood monocytes in SLE patients in remission was also less than that of the healthy controls. The IFN-γ-stimulated production of C4 in SLE patients in remission correlated with the peripheral blood CD4-positive cells. Less IFN-γ was produced by lymphocytes of SLE in remission than by those of healthy adults. We conclude that the serum C4 levels in SLE patients in remission reflect the degree of C4 consumption as well as the disease state, rather than genetic influences such as a C4A defect.

Serum C4 levels in patients with systemic lupus erythematosus in remission

Twenty-six patients with systemic lupus erythematosus (SLE) showing systemic lupus activity measure (SLAM) and SLE disease activity index (SLEDAI) scores ≤2, as well as a lower C4 concentration than the mean C4 levels of healthy controls, were selected to evaluate the C4 levels of SLE patients in remission. Serum complement (CH50), complement components (C4, C3, and B), complement split products (C4d, iC3b, and Bb), phenotypic expression of C4 allotype, C4 production by peripheral blood monocytes, peripheral blood lymphocyte subpopulation, and interferon-gamma (IFN-γ) production were examined. In patients with SLE in remission, the C4 consumption (C4d/C4) was found to increase, and this was considered to be the most important factor for determining the serum concentration of C4. However, the relevance of the C4 allotypic expression was minimal. The IFN-γ-stimulated production of C4 by peripheral blood monocytes in SLE patients in remission was also less than that of the healthy controls. The IFN-γ-stimulated production of C4 in SLE patients in remission correlated with the peripheral blood CD4-positive cells. Less IFN-γ was produced by lymphocytes of SLE in remission than by those of healthy adults. We conclude that the serum C4 levels in SLE patients in remission reflect the degree of C4 consumption as well as the disease state, rather than genetic influences such as a C4A defect.

Capillary deposition of complement split product C4d in renal allografts is associated with basement membrane injury in peritubular and glomerular capillaries: a contribution of humoral immunity to chronic allograft rejection.

Endothelial deposition of the complement split product C4d is an established marker of antibody-mediated acute renal allograft rejection. A contribution of alloantibody-dependent immune reactions to chronic rejection is under discussion. In this study, the association of immunohistochemically detected endothelial C4d deposition in peritubular capillaries (PTC) with morphologic features of chronic renal allograft injury was investigated in a large study cohort. C4d deposits in PTC were detected in 73 (34%) of 213 late allograft biopsies performed in 213 patients more than 12 mo after transplantation (median, 4.9 yr) because of chronic allograft dysfunction. Endothelial C4d deposition was found to be associated with chronic transplant glomerulopathy (CG) (P < 0.0001), with basement membrane multilayering in PTC (P = 0.01), and with an accumulation of mononuclear inflammatory cells in PTC (P < 0,001). Furthermore, C4d deposits in PTC (in biopsies with normal glomerular morphology) were associated with development of CG in follow-up biopsies. Other morphologic features of chronic allograft nephropathy (with exception of tubular atrophy) were not associated with C4d deposits in PTC. Analyses of previous and follow-up biopsies revealed that C4d deposits may occur de novo and may also disappear at any time after transplantation. In conclusion, the data suggest that complement activation in renal microvasculature, indicating humoral alloreactivity, contributes to chronic rejection characterized by chronic transplant glomerulopathy and basement membrane multilayering in PTC.

Capillary C4d deposition in kidney allografts: a specific marker of alloantibody-dependent graft injury.

Capillary deposition of the complement split product C4d has been discussed as a marker for antibody-mediated kidney allograft rejection. The relationship between C4d staining and posttransplant alloantibody detection remains to be thoroughly investigated, however. In this study, C4d staining in peritubular capillaries (PTC) and the incidence of alloantibody formation, as detected with sensitive techniques, were evaluated among a cohort of transplant recipients who had undergone biopsies and had not been selected for a specific histologic diagnosis. One hundred thirteen biopsies, obtained from 58 cadaveric kidney transplant recipients, were tested. Serum samples obtained at the time of biopsy were evaluated by flow cytometric crossmatch (FCXM) testing and FlowPRA (One Lambda, Inc., Canoga Park, CA) analysis of anti-HLA panel reactivity. Most biopsies with C4d deposits in PTC (C4d(PTC)(+), n = 21 of 24) were associated with positive posttransplant FCXM results (T and/or B cell FCXM) and/or > or =5% FlowPRA (anti-HLA class I and/or II) reactivity. Approximately 50% of the C4d(PTC)(-) biopsies were observed to be associated with donor-specific alloantibodies. Accordingly, high specificity (93%) but low sensitivity (31%) were calculated for capillary C4d staining (with FCXM testing as the standard method). For clinical evaluation, three patient groups were defined, i.e., a group of recipients with positive C4d staining in at least one allograft biopsy (C4d(PTC)(+), n = 16) and two C4d(PTC)(-) groups, which were discriminated on the basis of posttransplant FCXM results as C4d(PTC)(-)/FCXM(+) (n = 22) and C4d(PTC)(-)/FCXM(-) (n = 20) groups. Univariate analyses revealed significant differences between these groups with respect to serum creatinine levels at 12 mo [median, 2.83 mg/dl (interquartile range, 1.93 to 4.2 mg/dl) versus 1.78 mg/dl (1.47 to 2.24 mg/dl) versus 1.59 mg/dl (1.2 to 1.71 mg/dl), P < 0.001]. Of the five immunologic graft losses, four occurred in the C4d(PTC)(+) group and one occurred in the C4d(PTC)(-)/FCXM(+) group. In a multivariate analysis, C4d positivity was observed to have an independent predictive value for inferior 12-mo graft function (P = 0.02), whereas the observed moderate difference between C4d(PTC)(-)/FCXM(+) and C4d(PTC)(-)/FCXM(-) recipients did not achieve significance. In conclusion, these data demonstrate that positive C4d staining, which is an independent predictor of kidney graft dysfunction, represents a reliable specific marker for antibody-dependent graft injury.

Acute humoral renal allograft rejection.

In kidney transplantation, it is well established that donor-specific antibodies can cause substantial graft injury. Hyperacute rejection, now virtually eliminated by routine pretransplant cytotoxic crossmatch testing, represents the prototype of humoral rejection. However, there is now increasing evidence that alloantibody-mediated immune reactions may also cause acute rejection. Acute humoral rejection, which is frequently associated with severe graft dysfunction and immunologic graft loss, represents a particular diagnostic and therapeutic challenge. Reliable detection of antibody-mediated graft injury is required to govern the application of antihumoral therapeutic strategies. This review focuses on new approaches in the diagnosis and treatment of acute humoral rejection. Special attention is given to a novel diagnostic marker, the complement split product C4d.

Endothelial C4d deposition is associated with inferior kidney allograft outcome independently of cellular rejection.

Capillary deposition of complement split product C4d has been suggested to be a valuable marker for humoral rejection. In this retrospective study we evaluated the clinical impact of C4d deposition in renal allografts with special emphasis on associations between C4d staining patterns and histological features of acute rejection. One hundred and two allograft biopsies obtained from 61 kidney transplants (1-532 days after transplantation; median 14 days) were examined by immunohistochemistry on routine paraffin sections using a novel anti-C4d polyclonal antibody (C4dpAb). Fourty-two of 102 biopsies showed endothelial C4d deposits in peritubular capillaries (PTC). Histopathological analysis revealed a significantly lower frequency of positive C4d staining in biopsies with rather than in those without acute cellular rejection defined by the Banff grading schema (P<0.01). For clinical evaluation, patients were classified according to C4d staining in allografts (C4d(PTC) positive in at least one biopsy, n=31 vs C4d(PTC) negative in all biopsies, n=30). C4d(PTC) positive patients had significantly higher serum creatinine levels than C4d negative patients. Even in the absence of morphological evidence for rejection, differences in serum creatinine levels between C4d(PTC) positive and negative recipients were significant (6 months: 2.01+/-0.75 vs 1.41+/-0.27 mg/dl; 12 months: 1.95+/-0.60 vs 1.36+/- 0.34 mg/dl; 18 months: 1.98+/-0.50 vs 1.47+/-0.31 mg/dl; P<0.05). All patients with rejection resistant to conventional therapy (n=4) were in the C4d(PTC) positive subgroup. All recipients with panel reactive antibodies (PRA) >50% (n=8) were C4d(PTC) positive. Our data indicate that endothelial C4d deposition is associated with inferior graft outcome. We provide evidence that this immunohistochemical finding and its clinical impact are not associated with morphological signs of cellular rejection.

Activation of the complement system as an indicator of pyrogenic reaction to lipopolysaccharide (LPS)

The generation of biologically active complement split products through the direct reaction of microorganisms with complement proteins is one of the earliest events of the defence reaction in humans. Complement activation develops within minutes, which highly corresponds with the onset of a febrile reaction after exposure to pyrogens. The possibility of the use of complement activation in human plasma as an indicator of pyrogen contamination has been tested. Additionally, the co-stimulatory effect of complement activation on tumor necrosis factor-alpha (TNF-α) production by blood-separated macrophages exposed to lipopolysaccharide (LPS) has been demonstrated. As an indicator of complement activation in test samples, the concentration of the iC3b fragment was measured by using an ELISA system based on neoantigen formation. The 3-h exposure time has been identified as optimal for the test. The variability between iC3b concentrations in untreated control samples obtained from seven unrelated healthy donors was less than 10%, while after activation by 100 ng/ml LPS, it increased to 13%. The lower detection limit has been identified as 10 pg/ml LPS. As the complement test is not affected by drug–cell interactions or cell viability, the test can be used in situations where tested formulations contain active substances, which interfere with a cell-based test. We conclude that a test based on the detection of complement activation in human plasma should be considered as a valuable element of an in vitro pyrogenicity testing battery along with a cell-based assay.

ELISA for evaluating the incorporation of plasma derived complement split-products C3b/iC3b into solid-phase immune complexes

An ELISA that measures plasma derived complement (C) split-products C3b/iC3b deposited on solid-phase immune complexes during C activation is described. Plates are coated with BSA, anti-BSA and plasma is added. Deposited C3b/iC3b is then detected by biotinylated anti-C3c-antibodies, avidin-alkaline phosphatase and para-nitrophenylphosphate. A novel feature is that the assay measures residual C activation capacity rather than in vivo generated C activation products. The assay was applied to plasma from 250 healthy blood donors. No difference in activation capacity of either the alternative (AP) or classical pathway (CP) with regard to age or gender was demonstrated. The total coefficient of variation was <5.7%. The ELISA procedure was compared to a standard hemolytic complement CH 50 assay using plasma from 23 out-patients with systemic lupus erythematosus (SLE). There was a weak correlation between the two assays for both C pathways, but neither the ELISA nor the CH 50 assay showed any correlation with the diagnostic ACR-criteria for SLE. However, the capacity of the CP was significantly reduced in SLE out-patients compared to healthy blood donors ( P<0.0001).

C4d deposits in renal allografts are associated with inferior graft outcome

HERE IS INCREASING evidence for a pathogenetic role of humoral immunity in acute kidney allograft rejection. The complement split product C4d represents an attractive marker for antibody-mediated graft injury. Capillary C4d deposits in kidney allograft biopsies were found to be associated with the appearance of posttransplant anti-donor antibodies 1 and with poor graft outcome. 2 The objective of this retrospective study was to analyze the incidence and clinical relevance of C4d deposition in biopsies not selected for a specific type of allograft dysfunction with special emphasis on correlations between C4d staining patterns and histopathologic features of acute rejection.