Serial analysis of circulating immune complexes,complement, and antithymocyte globulin antibodies in heart transplant recipients

Thirteen heart transplant patients receiving heterologous antithymocyte globulin were examined serially for the presence of circulating immune complexes, activation of complement, and antibodies to antithymocyte globulin. Using three independent assays, all patients were found to develop immune complexes at some time following transplantation, mostly as a consequence of antithymocyte globulin administration and infection. Simultaneous analysis of endogenous complement levels revealed C3 and C4 hypocomplementaemia in 28 and 44% of the samples, respectively. However, no correlation was evident between immune complex or complement levels and cardiac rejection assessed histologically in 64 endomyocardial biopsies. Serial analysis of antibodies to equine and rabbit antithymocyte globulin by solid-phase radioimmunoassay showed that 8 of 10 patients had significantly elevated values. Evidence was also obtained which indicated a substantial degree of cross-reactivity between the antibodies to these heterologous proteins. The results demonstrate a complex interplay of immune reactants relating to transplantation, infection, and immunosuppression therapy.     

Double immunofluorescence studies of immunoglobulins, complement components and their control proteins in patients with IgA nephropathy

A study on double immunofluorescent staining of immunoglobulins, complement components, and their control proteins in renal tissues from patients with IgA nephropathy is described. Renal biopsy specimens were obtained from patients with IgA nephropathy. These biopsy specimens were stained with anti sera to human C1q, C4-binding protein, and beta 1H globulin by indirect immunofluorescent staining. These samples were then stained with rhodamine-conjugated anti human IgG, IgM, and IgA. Although C1q and C4-binding protein do not combine with IgA, they ubiquitously combine with IgG and/or IgM. beta 1H globulin also combine with IgG, IgM and/or IgA. It was demonstrated that the complement system activated in IgA nephropathy was via both alternative and classical pathways. The presence of C4-binding protein in glomeruli appeared to be a more sensitive indicator of classical pathway activation than the presence of C4.     

DOUBLE IMMUNOFLUORESCENCE STUDIES OF IMMUNOGLOBULINS, COMPLEMENT COMPONENTS AND THEIR CONTROL PROTEINS IN PATIENTS WITH IgA NEPHROPATHY

A study on double immunofluorescent staining of immunoglobulins, complement components, and their control proteins in renal tissues from patients with IgA nephropathy is described. Renal biopsy specimens were obtained from patients with IgA nephropathy. These biopsy specimens were stained with anti sera to human C1q, C4-binding protein, and β1H globulin by indirect immunofluorescent staining. These samples were then stained with rhodamine-conjugated anti human IgG, IgM, and IgA. Although C1q and C4-binding protein do not combine with IgA, they ubiquitously combine with IgG and/or IgM. β1H gobulin also combine with IgG, IgM and/or IgA. It was demonstrated that the complement system activated in IgA nephropathy was via both alternative and classical pathways. The presence of C4-binding protein in glomeruli appeared to be a more sensitive indicator of classical pathway activation than the presence of C4.     

Pathogenesis and significance of glomerular C4d deposition in lupus nephritis: activation of classical and lectin pathways

Immune complex-mediated complement activation through the classic pathway plays a key role in the pathogenesis of lupus nephritis (LN). C4d deposition in renal tissue reflects the prognosis of systemic lupus erythematosus (SLE). The aim of the current study is to investigate the pathogenesis and clinicopathologic significance of glomerular C4d deposition in LN. We retrospectively analyzed clinical and histopathological data of 20 SLE patients with renal biopsy-proven LN and 10 non-SLE renal biopsy samples as control. LN biopsies showed varying degrees of glomerular C4d staining associated with immune complex deposits, IgG (p = 0.015), C1q (p = 0.032) and C3 (p = 0.049). 7 LN biopsies had all of C4d, C1q and C3 deposits in their glomeruli, indicative of the activation of the classical pathway, whereas 2 LN biopsies had C4d and C3 deposits without accompanying C1q deposits, indicating the activation of the lectin pathway. Glomerular C4d deposition was correlated with the LN subtype (p < 0.001). In particular, a diffusely intense and coarsely granular pattern of C4d deposition in all glomeruli was detected in class V membranous LN. However, glomerular C4d deposition was correlated with neither disease activity of SLE nor histological activity and chronicity of LN. In conclusion, the activation of the lectin pathway as well as the classical pathway seems to play a crucial role in the pathogenesis of LN. Glomerular C4d staining could be helpful for diagnosing class V membranous LN, although glomerular C4d deposition does not reflect SLE disease activity and histological activity and chronicity.     

Classical complement activation as a footprint for murine and human antiphospholipid antibody-induced fetal loss

Recurrent miscarriage, fetal growth restriction and intrauterine fetal death are frequently occurring complications of pregnancy in patients with systemic lupus erythaematosus (SLE) and antiphospholipid syndrome (APS). Murine models show that complement activation plays a pivotal role in antiphospholipid antibody-mediated pregnancy morbidity, but the exact pathways of complement activation and their potential role in human pregnancy are insufficiently understood. We hypothesized that the classical pathway would play a major role in inducing fetal loss. Pregnant C57BL/6 mice and mice deficient in C1q and factor D were injected with antiphospholipid antibodies or normal human IgG. Mouse placentas were subsequently stained with an anti-C4 antibody and anti-normal human IgG to determine the presence of classical complement activation and IgG binding. Findings in mice were validated in 88 human placentae from 83 women (SLE and APS cases versus controls), which were immunohistochemically stained for C4d, C1q, properdin and MBL. Staining patterns were compared to pregnancy outcome. In murine placentae of mice pretreated with antiphospholipid antibodies, increased C4 deposition was observed, which was associated with adverse fetal outcome but not with IgG binding. In humans, diffuse C4d staining at the feto-maternal interface was present almost exclusively in patients with SLE and/or APS (p < 0.001) and was related to intrauterine fetal death (p = 0.03). Our data show that presence of C4d in murine and human placentae is strongly related to adverse fetal outcome in the setting of SLE and APS. The excessive deposition of C4d supports the concept of severe autoantibody-mediated injury at the fetal-maternal interface. We suggest C4d as a potential biomarker of autoantibody-mediated fetal loss in SLE and APS.     

Deposition of the lectin pathway of complement in renal biopsies of lupus nephritis patients

Background/aims

Lupus nephritis (LN) is one of the most serious manifestations of SLE occurring in 66–90% of these patients. The complement system is part of the innate immunity and modulator of inflammation and the adaptative immune response. Mannan-binding lectin (MBL) and Ficolin-2 (FCN-2) are important members of the lectin pathway of complement activation. Despite the significant participation of complement in the pathogenesis of the LN, there are few reports demonstrating “in situ” deposition of complement components in renal biopsy specimens in this disorder. The present study investigated the deposition of complement components in kidney specimens of LN patients.

Methods

Renal biopsies of 11 patients with SLE and LN were evaluated for immunofluorescence staining for IgG, IgA, IgM, C3, and C1q. Additionally, MBL, FCN-2 and C5b-9 were researched using monoclonal antibodies.

Results

All the biopsies were positive for IgG, C3, and C1q, eight were positive IgM and five had IgA deposition in glomerular tissue. The terminal complex of complement C5b9 was positive in all cases, MBL in nine (82%) cases; seven (63.6%) of them presenting concomitantly FCN-2 deposition. Patients presenting MBL deposition had higher mean of urinary proteins (9.0 g/day) than patients with negative MBL deposition (mean of 2.3 g/day).

Conclusions

In this study, we demonstrated in situ the participation of complement in the renal injury, including MBL and FCN-2 of the lectin pathway; also the strong role of C5b-9 in the pathogenesis of LN.     

Vascular deposition of complement-split products in kidney allografts with cell-mediated rejection.

Complement activation in 73 renal transplant biopsies was investigated by indirect immunoperoxidase staining using MoAbs reactive with complement-split products. Intense deposition of complement fragments C4d and C3d in peritubular capillaries, indicating activation of the classical pathway, could be detected in the majority of transplanted kidneys with cell-mediated rejections. Abundant deposition of complement-split products was observed in 22 early biopsies from patients with high 'immunological risk' (i.e. previous, rejected transplants and/or circulating antibodies against HLA-antigens). Despite negative results in the crossmatch before transplantation and paucity of immunoglobulins in transplant biopsies, antibodies directed against endothelial cell antigens should be considered as a possible cause of classical complement activation.     

The role of complement activation in autoimmune liver disease

IntroductionThe complement system, an essential part of the innate immune system, is involved in various autoimmune diseases. Activation of the complement system by autoantibodies results in immune activation and tissue damage. At the moment little is known about the role of the complement system in autoimmune liver disease, including primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH). Since inhibition of the complement system is currently being tested in several autoimmune diseases as a therapeutic option, its role in autoimmune liver disease requires further clarification.MethodsA review of the literature was performed on studies investigating complement activation in PBC, PSC and AIH. Since data on AIH were lacking immunohistochemical staining for IgG, C1q, C3d, C4d and C5b9 was performed on liver tissue of nine AIH patients, two healthy controls and one positive control (acute liver failure caused by paracetamol intoxication).ResultsImmunohistochemical analysis in AIH revealed increased production of C3 and C4 by hepatocytes. Despite a strong staining for IgG in the immune infiltrate in AIH, C3d, C4d and C5b9 deposition was only present in one AIH patient and the deposition was restricted to the interface between portal tracts and liver parenchyma. No deposition was found in all other AIH patients or healthy controls.Literature review showed raised plasma C3 and C4 levels in AIH, PBC and PSC patients compared to healthy controls. For PBC and PSC no complement depositions at the bile ducts were reported.Conclusion and discussionAlthough complement is involved in various autoimmune diseases, the role of complement in autoimmune liver disease seems limited. Therefore it is unlikely that complement inhibition will become a novel treatment option for these diseases.     

Failure to detect deposition of complement and immunoglobulin in allergen-induced late-phase skin reaction in atopic subjects.

There still remains some controversy regarding the possible role of immune complexes in the pathogenesis of the late-phase skin reaction (LPSR). To assess this, skin biopsies were obtained from LPSR induced in atopic human subjects 6, 24 and 48 h after allergen challenge. Cryostat sections were stained by direct immunofluorescence for the presence of fibrinogen, immunoglobulin classes IgM and IgG and for the complement components C1q and C3c. Complement components were observed in only two of the 29 biopsies studied. In both instances, only C3c was detected. One of these subjects also had unequivocal IgG staining at 6 h. IgM staining was detected in two out of 10 subjects at 6 h but no significant deposition of immunoglobulins could be found at 24 or 48 h. Fibrinogen deposition was observed in about half of the biopsies at each time-point. This study suggests that substantial complement and immunoglobulin deposition are not overt features of the allergen-induced LPSR, although the presence of small amounts of immune complexes, below the sensitivity of the method employed cannot be excluded. Fibrin deposition occurs in the LPSR but does not appear to be a prerequisite for LPSR development.     

Re-examination of sinusoidal deposition of complement 4d in liver allografts: experience from a single institution

Complement 4d (C4d) is a marker of complement activation that has been used to evaluate humoral rejection in renal and heart allografts. Studies suggested a role for C4d detection in liver allografts in diagnosing acute cellular and humoral rejection but none correlated this with the pre-transplant liver disease. Our study analyzed the association of C4d deposition in liver allografts with the pre-transplant liver disease. C4d deposition was evaluated by indirect immunofluorescence and correlated with lymphocytotoxic crossmatch results, post-transplant clinicopathological diagnosis and type of pre-transplant liver disease. Allograft biopsies were classified by the native liver disease. After excluding 20 patients with rare liver diseases; C4d deposition was evaluated in 506 biopsies from 310 patients including 25 with PSC, 198 with viral hepatitis and 87 with other diseases. C4d immunereactivity distribution was not different among biopsies from patients with different lymphocytotoxic crossmatch results. Sinusoidal C4d deposition was noted in 11.9% of biopsies and 15.2% of patients (in one or more biopsies). 26% (9/35) of biopsies from patients with PSC had sinusoidal C4d deposition; more frequently than from patients with viral hepatitis 12% (43/348) (p=0.04) and other liver diseases 7% (8/123) (p=0.005). In conclusion, C4d deposition in liver allografts is independent of the crossmatch results. It occurs with a variety of pathologic abnormalities and underlying liver diseases; but is more frequent in patients with PSC. This suggests that mechanisms other than allo-immunity activate complement. The mechanisms and clinical significance of C4d deposition in liver allografts in patients with PSC remain to be determined.     

Deposition of complement activation products on plastic-adsorbed immunoglobulins. A simple ELISA technique for the detection of defined complement deficiencies

The activation of complement components in human serum has been studied using immunoglobulins adsorbed to microtiter plates. The sequential deposition of complement fragments was detected by a series of mono- and polyclonal antibodies in an indirect enzyme-linked immunosorbent assay (ELISA). Antibodies against C1q, C1s, C4b/d, C3b/d, factor B, C5b-9 membrane attack complex (MAC), the regulatory complement proteins C4 binding protein (C4bp) and properdin were reactive. Several lines of evidence suggest that complement activation was via the classical pathway: (1) complement activation was highly isotype-restricted with regard to the adsorbed Igs (human IgG1 and IgG3 as well as mouse IgM, IgG2a and IgG2b isotypes are strong activators in contrast to human IgG2, IgG4, IgA and mouse IgG1); (2) Ca2+ depletion, heat treatment (56 degrees C for 45 min), incubation with 0.5 M KSCN or heat-aggregated immunoglobulins (aggIgG) abrogated serum activity; (3) complement deficient sera (C1q def', C2 def', C6 def' human sera; C2 def', C4 def' guinea pig sera) showed impaired deposition of the complement components that follow the missing component in the cascade of activation. In a clinical study sera from patients with systemic lupus erythematosus (SLE) were investigated in order to measure the effect of hypocomplementemia due to complement consumption. The results obtained suggest that this new and simple assay is well suited for (1) the detection of various inherited complement deficiencies, (2) the semiquantitative evaluation of sera with decreased complement levels, (3) a more detailed study of complement components bound to a solid phase.     

Significance of C4d staining in ABO-identical/compatible liver transplantation.

Complement degradation product C4d has become an important marker of humoral or antibody-mediated rejection in renal and heart allograft biopsies. Although there have been several reports on the detection of C4d in liver allografts, the significance of C4d in liver transplantation and its relationship with humoral rejection are still not clear. We investigated the frequency and pattern of C4d staining in liver allograft biopsies with reference to preoperative lymphocyte crossmatch tests, which detect donor-reactive lymphocyte antibody. Survival rates at 5 years were 77% for crossmatch-negative patients and 53% for crossmatch-positive patients (P=0.009). In crossmatch-negative patients, reproducible positive staining was obtained in 28 of 86 (33%) biopsies taken within 90 days after transplantation and 33 of 96 (34%) biopsies 90 days or after transplantation. Most C4d staining was observed in the portal areas, and no clear correlation was observed between C4d positivity and histological diagnosis. In crossmatch-positive patients, 9 of 11 (82%) biopsies showed positivity for C4d. C4d stained perivenular areas as well as portal areas. Histology of crossmatch-positive patients included acute rejection and cholangitis, but did not include periportal changes that were seen in humoral rejection in ABO-incompatible liver transplantation. In summary, focal C4d deposition was seen in various types of liver allograft injury and had little clinical impact on crossmatch-negative patients, but extensive C4d staining in crossmatch-positive patients may be associated with humoral rejection and poor graft survival.     

急性排斥移植肾组织嗜酸性粒细胞浸润及C4d沉积

目的：通过观察移植肾PTC C4d沉积和嗜酸性粒细胞浸润与AHR的关系,探讨两者之间的相关性及其在诊断急性体液排斥中的作用。方法：采用间接免疫荧光法检测移植肾组织C4d沉积。26例移植肾PTC C4d弥漫性强阳性染色患者为C4d阳性组,而30例移植肾PTC C4d阴性患者为对照组。光镜下记数移植肾组织嗜酸性粒细胞数。结果：（1）26例C4d阳性患者中经病理诊断为AHR为13例,AHR＋ACR为8例,ACR1为3例,ACR2为2例。30例C4d阴性患者中,AHR为1例,AHR＋ACR为2例,ACR1为21例,ACR2为6例。与C4d阴性组相比,C4d阳性组难治性排斥患者较多,常需进一步干预性措施,且较多的患者对抗排斥治疗无反应,移植肾失功率高（7/26）。（2）C4d阳性组移植肾浸润嗜酸性粒细胞数亦明显高于C4d阴性组,两组相比差异显著（P〈0.01）。C4d阳性无细胞性排斥组移植肾间质浸润嗜酸性粒细胞数与C4d阳性＋ACR组相比差异无显著性（P〉0.05）。C4d阳性ACR组移植肾间质浸润嗜酸性粒细胞数明显高于C4d阴性ACR组（P〈0.05）。结论：移植肾PTC C4d沉积与嗜酸性粒细胞浸润密切相关,两者在肾移植AHR诊断过程中起重要作用。     

Flow cytometry based detection of HLA alloantibody mediated classical complement activation

Complement-dependent cytotoxicity (CDC) panel reactive antibody (PRA) testing is used to assess recipient presensitization and post-transplant alloantibody formation in transplant recipients. However, CDC test results can be affected by false-positive reactions brought about by autoantibodies or antilymphocyte reagents. As an alternative to the CDC-PRA assay, detection of HLA alloantibodies using HLA antigen-coated microbeads (FlowPRA test) was recently established. FlowPRA testing, however, does not distinguish between (presumably more harmful) complement-fixing and noncomplement-fixing alloantibodies. In this study, we established a novel assay allowing flow cytometric detection of HLA alloantibody dependent classical complement activation using the FlowPRA test. For the detection of complement activation, FlowPRA beads were incubated with sera from highly sensitized dialysis patients (CDC-PRA reactivity >60%) and then stained for C4 (C4d, C4c) and C3 (C3d, C3c) fragments, as well as C1q deposition using indirect immunofluorescence. We demonstrate alloantibody induced induction of C4 fragment, and in parallel C1q deposition to HLA class I or class II beads. As shown by immunoblotting, C4 staining was not due to the presence of preformed C4 fragment-IgG/M complexes. Indeed, C4 fragment deposition in our in vitro system was demonstrated to result from de novo complement activation. First, inactivation of C4 by treatment of sera with methylamine, which inhibits cleavage of the internal thioester, completely abolished C4 fragment deposition. Second, C4 fragment deposition was not observed in the evaluation of C4-free immunoadsorption eluates obtained from highly sensitized dialysis patients. After supplementation with complement, however, eluates induced C4 deposition. Deposition of C4 split products and C1q was temperature-dependent with maximum binding after incubation at 4 degrees C for 60 min. In contrast, maximum C3 fragment deposition was found at 37 degrees C. At this temperature, C3 deposition occurred in an alloantibody and C4-independent fashion, presumably as a result of alternative complement activation. In summary, we describe a novel cell-independent and easy-to-perform PRA test that permits flow cytometry based detection of alloantibody induced classical complement activation. Future studies will have to evaluate its possible relevance as an alternative to CDC-PRA testing in clinical transplantation.     

Complement deposition in renal allografts with early malfunction

Among patients with early severe impairment of renal allograft function we have previously identified a group displaying isolated deposition of complement factor C3 in glomeruli. Here we studied the pattern of complement deposition more extensively in allograft biopsies from five patients using an immunofluorescence technique. We found a prominent deposition of C3c, C3d and C4d antigens in the glomerular capillary walls, and a positive reaction to vitronectin (S-protein), but only trace amounts of the complement factor C9 neoepitope. Clq, C4c, C3a, iC3b, factor B, properdin, immunoglobulins IgG, IgA or IgM were not found in glomeruli or in any other cortical structure. These findings indicate that most of the demonstrated glomerular C3 consists of C3b and/or C3c/C3d molecules. By immunoelectron microscopy the C3 antigen was found within the glomerular basement membrane. Our findings indicate that there is a mechanism of complement activation involving the early steps of the classical pathway, despite the lack of demonstrable immunoglobulins in the tissue. In analogy with similar reactions described recently in heart allografts, we suggest that this may be a manifestation of a humoral rejection, possibly mediated by a low titer of circulating antibodies directed against endothelial surface antigens, presumed to be the initial step leading to complement activation.     

Cryoprecipitate of patients with cryoglobulinemic glomerulonephritis contains molecules of the lectin complement pathway

Serological and histological studies were carried out to explore the role of the lectin complement pathway in the pathogenesis of cryoglobulinemic glomerulonephritis. Sixteen patients with mixed cryoglobulinemia type II with glomerulonephritis (GN) were enrolled. All cases had hepatitis C virus (HCV) infection. The serum concentration of mannose-binding lectin (MBL) was significantly higher in the GN patients than in the normal controls according to ELISA (P < 0.01). IgG, IgM, C1q, C4d, HCV envelope antigen, MBL, and MBL-associated serine protease-1 (MASP-1) could be visualized in the cryoprecipitate of the 16 patients by Dot blot assay. Renal biopsy specimens obtained from 3 patients were examined by immunohistochemistry, and the glomeruli strongly stained for IgG, IgM, MBL, MASP-1, C4d, C3c, and C3d in a fringe-like pattern. The pattern of HCV constituent deposition was partially fringe-like. The complement profiles of the 16 cases were distinctive; briefly, the serum levels of C1q, C2, and C3 were reduced, although the levels of circulating regulatory proteins (C1-inhibitor, factor H, and factor I) were in the normal range. The serum C4 level was significantly reduced. These results indicate that immune complex formation involves molecules of the lectin pathway and leads to organ damage in cryoglobulinemic glomerulonephritis.     

Glomerular C4d deposition indicates in situ classic complement pathway activation,but is not a marker for lupus nephritis activity

This study was designed to evaluate whether glomerular C4d deposition may be a useful marker of lupus nephritis activity. Twenty-one patients diagnosed as having lupus nephritis (WHO class III: 4 cases; IV: 12 cases; V: 5 cases) were included. Mean patient age was 29.3 +/- 13.5 years (range: 7-55 years). The presence and intensity of glomerular C4d deposition were compared with the corresponding histologic activity index for each case. Immunofluorescence for C4d showed diffusely granular staining along glomerular capillary loops, in all cases examined (1+, in 8 cases; 2+, in 7 cases; 3+, in 6 cases). In eight cases, C4d deposition was found in the absence of capillary or mesangial C4 deposits. Moreover, the intensity of C4d deposits correlated with those of capillary IgG, IgA, C4, C1q, and fibrinogen deposits. However, C4d staining intensity did not correlate with the lupus nephritis activity index. Although glomerular capillary C4d deposition is a sensitive marker of classic complement pathway activation, it is not a sensitive marker for active lupus nephritis.     

Antithymocyte globulin treatment in patients with aplastic anemia: a prospective randomized trial

We evaluated the efficacy of antithymocyte globulin for the treatment of moderate to severe aplastic anemia in a randomized controlled study. Eleven of 21 patients initially randomized to receive antithymocyte globulin (given intravenously on eight consecutive days) had sustained improvement in hematopoiesis within three months of treatment; none of 21 control patients who received supportive care alone improved (P = 0.0005). Six of 12 control patients who subsequently received antithymocyte globulin improved. Responders had gradual improvement in hematopoiesis, but none recovered completely normal peripheral-blood counts. The severity of bone-marrow failure, age, cause of aplastic anemia, and transfusion history had no apparent bearing on treatment outcome. The interval from diagnosis to antithymocyte globulin treatment correlated inversely with the chance of a treatment response, although this correlation was not statistically significant. These data indicate that antithymocyte globulin is effective in improving hematopoiesis in some patients with aplastic anemia.     

Immunoperoxidase staining for C4d on paraffin-embedded tissue in cardiac allograft endomyocardial biopsies: comparison to frozen tissue immunofluorescence.

C4d deposition in microvasculature is a marker for humoral rejection. The authors compared a recently developed C4d immunoperoxidase (IP) method for paraffin-embedded tissue to immunofluorescence (IF) of frozen tissue. Of 315 frozen endomyocardial biopsies with IF staining for C4d, 280 were negative and 35 were positive. Negative controls were 17 negative biopsies and 11 biopsies with myocyte necrosis. The extent of IP and IF staining was graded as 0 to 3+. Staining intensity and the number and type of positive vessels were recorded. Staining patterns in Quilty lesions (QL) and foci of acute cellular rejection (ACR) were also evaluated. In 34 biopsies with sufficient tissue, IP criteria of 2+/3+, or more than 10 to 20 positive vessels per 10 high-power fields detected 25.0% (1/4), 18.2% (2/11), and 84.2% (16/19) of 1+, 2+, and 3+ IF-positive biopsies, respectively, without false positives. Considering C4d IF 3+ as positive resulted in 84.2% (16/19) sensitivity and 93.0% specificity (40/43). Intensely stained capillaries predominated in six of seven biopsies when more than 100 capillaries per 10 high-power fields were positive. Seventy percent (7/10) of IP 2+ and 3+ biopsies showed positive capillaries in QLs, while 36.4% (4/11) of IP 1+ and negative biopsies did. All eight IP 2+/3+ biopsies showed positive capillaries in ACR foci, while 25.0% (1/4) of IP-negative biopsies did. Capillary staining in QLs and areas of ACR reflects overall C4d deposition. In conclusion, IP staining of 2+/3+ is highly sensitive and specific for C4d positivity. The authors recommend considering 2+ and 3+ as positive staining when using the IP technique.     

Evaluation of in vivo immune complex formation and complement activation in patients receiving intravenous streptokinase.

The usefulness of several different methods for detecting immune complex formation and complement activation in the circulation were applied to samples from patients receiving intravenous Streptokinase therapy for myocardial infarction. Streptokinase is a foreign antigen and can cause immune reactions. We collected samples from 13 patients, before Streptokinase administration (baseline), at the end of infusion (1 h), 12 h later and on day 7. We measured IgG containing immune complexes (IgG-IC), free C3d and antibodies to Streptokinase by ELISA, and CR1, C3d and C4d on erythrocytes by flow cytometric assay. Antibodies to Streptokinase are common, as all but two of the patients had measurable antibody levels. During Streptokinase treatment there was a drop in antibody levels, most prominent in those patients who had high baseline levels. At the same time increased levels of free C3d and erythrocyte-bound C3d were observed. After 12 h free C3d was usually back to baseline level, but C3d on erythrocytes was still raised. These data indicate the formation of Streptokinase immune complexes in patients with high Streptokinase antibody levels, and show that these complexes are cleared rapidly from the circulation, leaving more persistent signs of complement activation. We conclude that free C3d is a good indicator of ongoing complement activation, whereas C3d on erythrocytes indicates that complement activation has recently taken place.