Effect Of Complement Inhibition Research Articles

Novel uses of complement inhibitors in myasthenia gravis-Two case reports.

Myasthenia gravis (MG) is a rare, life-threatening immune-related adverse effect (irAE) of immune checkpoint inhibitor (ICI) treatment. C5-complement inhibitors are effective treatments for acetylcholine receptor antibody (AChR ab) positive generalized MG. We describe the use of eculizumab/ravulizumab in two patients with MGreceiving concomitant pembrolizumab. This was a retrospective review of two medical records. Patient 1: An 80-year-old male with recurrent, non-muscle invasive transitional cell carcinoma of the bladder developed ICI-induced AChR ab positive MG (ICI-MG), myositis, and myocarditis 2 weeks after the first dose of pembrolizumab. Myositis responded to corticosteroids. MG responded to eculizumab, followed by ravulizumab. He died of metastatic cancer 8 months later. Patient 2: A 58-year-old male had refractory thymoma-associated AChR ab-positive MG, which responded to eculizumab. He developed metastatic Merkel cell cancer necessitating pembrolizumab. MG remained stable on eculizumab. He had no irAEs for 22 months, with positron emission tomographic resolution of cancer. He then developed mild, indolent retinal vasculitis, which responded to prednisone. Discontinuation of pembrolizumab for 5 months resulted in cancer recurrence; pembrolizumab was resumed with peri-infusion pulse prednisone. MG remained stable and he continues eculizumab. In the first patient, eculizumab, followed by ravulizumab, improved ICI-MG. In the second patient, eculizumab treatment may have had a prophylactic effect on the development of ICI-induced irAEs. The effect of complement inhibition on cancer outcomes of ICI therapy is unknown. A possible biologic basis for complement inhibitors in reducing irAEs of ICI, especially in the presence of underlying autoimmune disease, merits evaluation.

The potential role of complement alternative pathway activation in hypertensive renal damage.

Hypertensive renal damage is a common secondary kidney disease caused by poor control of blood pressure. Recent evidence has revealed abnormal activation of the complement alternative pathway (AP) in hypertensive patients and animal models and that this phenomenon is related to hypertensive renal damage. Conditions in the setting of hypertension, including high renin concentration, reduced binding of factor H to the glomerular basement membrane, and abnormal local synthesis of complement proteins, potentially promote the AP activation in the kidney. The products of the AP activation promote the phenotypic transition of mesangial cells and tubular cells, attack endothelial cells and recruit immunocytes to worsen hypertensive renal damage. The effects of complement inhibition on hypertensive renal damage are contradictory. Although clinical data support the use of C5 monoclonal antibody in malignant hypertension, pharmacological inhibition in hypertensive animals provides little benefit to kidney function. Therefore, the role of the complement AP in the pathogenesis of hypertensive renal damage and the value of complement inhibition in hypertensive renal damage treatment must be further explored.

Complement Regulates the Procoagulant Effects of HIT Immune Complexes

Heparin induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by ultra-large immune complexes (ULICs) containing IgG antibodies bound to multivalent complexes of platelet factor 4 (PF4) and heparin (H). HIT ULICs activate cellular FcγIIA receptors that initiate diverse cellular effector functions including neutrophil degranulation and monocyte expression of tissue factor (TF). Previous studies have shown that HIT ULICs also potently activate complement through the classical pathway (Cines et al., 1980). Whether complement activation contributes to FcγRIIA-dependent prothrombotic pathways has not been addressed in detail. In studies that follow, we describe: 1) robust complement activation by HIT ULICs in plasma and whole blood (WB), 2) cell-surface deposition of complement and IgG triggered by HIT ULICs, 3) complement-dependent neutrophil degranulation and monocyte TF expression, 4) efficacy of proximal, but not terminal, pathway inhibition in regulating monocyte TF expression, and 5) deposition of complement in thrombi formed in "HIT mice" that generate ULICs containing KKO, a HIT-like monoclonal antibody (Arepally et al., 2000). Consistent with prior studies showing involvement of the classical pathway in HIT (Cines et al., 1980), we observed that binding of C1q induced marked enlargement of HIT ULICs in buffer assessed by dynamic light scattering as well as in plasma using confocal microscopy (data not shown). To assess complement activation by HIT ULICs, we incubated WB and plasma with PF4 (25 µg/mL) ± heparin (1 U/mL) in the presence of KKO (or isotype, "ISO"; 50 µg/mL) or HIT IgG (or control IgG, "CON"; 500 µg/mL) and measured C3c with a capture immunoassay as previously described (Khandelwal et al., 2018). KKO (Figure 1A) or HIT ULICs (n=3; HIT1-3, Figure 1B), showed robust generation of C3c in the presence of PF4/heparin, but not antigens alone or with control IgG (ISO/CON). Complement activation by HIT ULICs leads to downstream generation of C5a and formation of sC5b-9 (data not shown). Pre-incubation of plasma or WB with a variety of classical pathway inhibitors, including a C1r inhibitor derived from Borrelia burgdorferi (BBK 32), C1 esterase inhibitor (Berinert, CSL Behring) and anti-C1q antibody (α-C1q Ab; Annexon Biosciences) inhibited C3c generation by KKO ULICs (p <0.001), whereas inhibitors of the alternative pathway (anti-properdin antibody) or C5 inhibitor (α-C5 Ab; Eculizumab, Alexion Pharmaceuticals) did not (data not shown). Incubation of WB with KKO or HIT ULICs, but not ISO or CON IgG, markedly increased deposition of C3 and IgG on neutrophils, monocytes and B cells (data not shown) and lead to cell activation assessed by neutrophil degranulation (MMP9 release) and monocyte TF expression (data not shown). To examine the contribution of complement activation in monocyte TF expression, WB was pre-incubated with α-C1q, α-C5 or IV.3 (a monoclonal antibody to FcγRIIA) or isotype controls prior to addition of HIT ULICs. As shown in Figure 2, the classical pathway inhibitor, α-C1q Ab markedly diminished TF expression (about 70% reduction; p<0.001 vPF4/H/ KKO), as did IV.3 (about 85% reduction; p<0.001 vPF4/H/ KKO) but not α-C5 Ab or ISO antibodies, demonstrating: 1) FcγRIIA independent mechanism of monocyte TF expression and 2) a requirement for proximal rather than terminal complement pathway components in the induction of monocyte TF. We next asked if complement activation facilitates binding of ULICs and promotes subsequent ULIC engagement of FcγRIIA. To examine complement dependent binding of HIT ULICs, we incubated WB with α-C1q Ab prior to addition of KKO ULICs and measured ULIC binding to monocytes and TF expression. As shown in Figure 3, classical pathway inhibition markedly reduced cell-surface IgG (Figure 3A) and monocyte TF expression (Figure 3B). The effects of complement inhibition could not be overcome with increasing amounts of KKO IgG (2-4 fold excess). We observed significant co-localization of complement with KKO ULICs in a cremaster-laser injury model in "HIT mice" and in in situ thrombi formed in uninjured vessels (data not shown). Together, these studies demonstrate an independent role for complement activation in regulating the binding and procoagulant effects of HIT ULICs and identify new non-anticoagulant therapeutic targets that could improve clinical outcomes in this otherwise potentially devastating thrombotic disorder. Disclosures Arepally: Novartis: Consultancy; Alexion: Other; Annexon Biosciences: Consultancy, Other; Veralox Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biokit: Consultancy, Patents & Royalties; Apotex: Consultancy, Research Funding.

Effect of Complement Inhibition on Lymphocytes in Treatment Refractory Myasthenia Gravis Patients (4524)

Effect of Complement Inhibition on Lymphocytes in Treatment Refractory Myasthenia Gravis Patients (4524)

Phagocytosis of live and dead Escherichia coli and Staphylococcus aureus in human whole blood is markedly reduced by combined inhibition of C5aR1 and CD14

Sepsis is a dysregulated host response to infection. The aim of this study was to investigate the effects of complement- and CD14 inhibition on phagocytosis of live and dead Gram-negative and Gram-positive bacteria in human whole blood. Lepirudin-anticoagulated blood was incubated with live or dead E. coli or S. aureus at 37 °C for 120 min with or without the C5aR1 antagonist PMX53 and/or anti-CD14. Granulocyte and monocyte phagocytosis were measured by flow cytometry, and five plasma cytokines by multiplex, yielding a total of 28 mediators of inflammation tested for. 16/28 conditions were reduced by PMX53, 7/28 by anti-CD14, and 24/28 by combined PMX53 and CD14 inhibition. The effect of complement inhibition was quantitatively more pronounced, in particular for the responses to S. aureus. The effect of anti-CD14 was modest, except for a marked reduction in INF-β. The responses to live and dead S. aureus were equally inhibited, whereas the responses to live E. coli were inhibited less than those to dead E. coli. C5aR1 inhibited phagocytosis-induced inflammation by live and dead E. coli and S. aureus. CD14 blockade potentiated the effect of C5aR1 blockade, thus attenuating inflammation.

Staphylococcus aureus‐induced complement activation promotes tissue factor‐mediated coagulation

Background There is extensive cross-talk between the complement system, the Toll-like receptors (TLRs), and hemostasis. Consumptive coagulopathy is a hallmark of sepsis, and is often mediated through increased tissue factor (TF) expression. Objectives To study the relative roles of complement, TLRs and TF in Staphylococcus aureus-induced coagulation. Methods Lepirudin-anticoagulated human whole blood was incubated with the three S. aureus strains Cowan, Wood, and Newman. C3 was inhibited with compstatin, C5 with eculizumab, C5a receptor 1 (C5aR1) and activated factor XII with peptide inhibitors, CD14, TLR2 and TF with neutralizing antibodies, and TLR4 with eritoran. Complement activation was measured by ELISA. Coagulation was measured according to prothrombin fragment 1 + 2 (PTF1 + 2 ) determined with ELISA, and TF mRNA, monocyte surface expression and functional activity were measured with quantitative PCR, flow cytometry, and ELISA, respectively. Results All three strains generated substantial and statistically significant amounts of C5a, terminal complement complex, PTF1 + 2 , and TF mRNA, and showed substantial TF surface expression on monocytes and TF functional activity. Inhibition of C5 cleavage most efficiently and significantly inhibited all six markers in strains Cowan and Wood, and five markers in Newman. The effect of complement inhibition was shown to be completely dependent on C5aR1. The C5 blocking effect was equally potentiated when combined with blocking of CD14 or TLR2, but not TLR4. TF blocking significantly reduced PTF1 + 2 levels to baseline levels. Conclusions S. aureus-induced coagulation in human whole blood was mainly attributable to C5a-induced mRNA upregulation, monocyte TF expression, and plasma TF activity, thus underscoring complement as a key player in S. aureus-induced coagulation.

OR12 Complement-binding donor-specific anti-HLA antibodies predict the response to complement-targeting therapy: A multicenter international study

Aim We investigated whether circulating complement-binding donor-specific anti-HLA antibodies (DSA) influence response to complement-targeting therapy. Methods We included 116 kidney recipients transplanted between 2011 and 2014 in 10 referral centers (US and Europe) from two prospective clinical trials investigating the effect of complement inhibition for rejection prophylaxis in kidney recipients with anti-HLA DSAs at transplantation (NCT01567085 and 01399593). They received either Eculizumab in the first nine weeks post-transplantation (N = 52) or standard of care (SOC) consisting in plasma exchange and intravenous immunoglobulin (N = 64). All patients were screened for C1q-binding DSAs using SAB in sera collected at transplantation and underwent allograft biopsy at day 14 to assess histological characteristics and allograft gene expression (microarray). We also compared the 3-month incidence of biopsy-proven antibody-mediated rejection (AMR) according to therapy. Results Patient baseline characteristics were similar between the 2 treatment groups. 69 (59%) patients had pre-transplant C1q-binding DSA (32 receiving SOC and 37 receiving Eculizumab) and 47 (41%) had non-C1q-binding DSA (32 receiving SOC and 15 receiving Eculizumab). In patients with C1q-binding DSAs, compared with patients receiving SOC, Eculizumab was associated with decreased glomerulitis (P = 0.001), peritubular capillaritis (P Conclusions Circulating complement-activating anti-HLA DSAs identify kidney transplant recipients who respond to complement inhibition.

The effect of complement inhibition on erythrocyte destruction in AIHA

To determine differences in the provision of Medicare services based on physician gender in the United States.Participants included all 2013 Medicare fee-for-service physicians and their patients, a population that is predominantly older than 65 years. The 2013 Medicare Provider Utilization and Payment Data for services rendered between January 1, 2013, and December 31, 2013, were combined with the 2015 Physician Compare National Downloadable files and 2015 Berenson-Eggers Type of Service classification files. Total fee-for-service Medicare payments and Healthcare Common Procedure Coding System procedure codes for all fee-for-service beneficiaries were aggregated according to physician gender, specialty, years since medical school graduation, and type of service classifications.Excluding drug reimbursement, the mean total Medicare payments per female physician, compared with those for male physicians, were 41% in surgical specialties, 72% in hospital-based specialties, and 55% across all specialties (P<.001). The mean overall number of unique beneficiary visits per female physician was 59% of that for male physicians (P<.001). By using the Berenson-Eggers Type of Service classification, procedures and other services by female physicians were of 54% lower overall average intensity (allowed payments/number of unique patients) compared with those of male physicians. These differences persisted irrespective of years since medical school graduation (P<.001).Female physicians had smaller average total Medicare payments and fewer unique beneficiary visits than male physicians in the care of fee-for-service Medicare beneficiaries in 2013. The differences persisted across specialty types and years in practice. These data can identify variation but cannot determine causation or explain the reasons behind gender differences. These findings suggest, but do not prove, that female physician Medicare payments are lower due to different practice patterns, consisting of fewer patients cared for and lower intensity of care.

Effect of Complement Inhibition By Anti-C5 (Eculizumab) or a Small Molecule Inhibitor of Factor D (ACH-4471) on Survival of Meningococci in Blood from Vaccinated Adults

Abstract Background. Complement pathways are important targets for treatment of paroxysmal nocturnalhemoglobinuria (PNH) and autoimmune diseases. Because complement is essential in host defense against Neisseria, patients treated with complement inhibitors are at increased risk of life-threatening meningococcal disease. Meningococcal vaccination is recommended. However, the ability of vaccine-induced antibodies to confer protection when different complement pathways are blocked is poorly understood. Inhibiting formation of the membrane attack complex, C5b-C9, blocks serum bactericidal activity (SBA). In the absence of SBA, antibodies can prevent meningococcal disease byopsonophagocytosis (OPA) by binding of antibody to bacteria and activation of C4b and C3b via the classical pathway (CP). When antibody is limited, or the antigenic target is sparse, amplification of C3b deposition via the alternative pathway (AP) is required. OPA killing also depends on activation of phagocytic cells and up-regulation of Fc- and CR3 receptors, which is stimulated by C5a. In this study, we used a whole blood meningococcal killing assay to investigate blocking terminal pathway components C5 or C7 on bacterial survival. We also investigated the effect of blocking the AP using a small molecule factor D inhibitor. Methods. Bloodanticoagulated withlepirudin was obtained from five adults previously immunized with a meningococcal polysaccharide or conjugate vaccine. Four subjects also had completed serogroup B vaccination within 10 months. At time 0, ~5000cfu/ml of serogroup B or C bacteria were added to blood. Meningococcal killing was measured after incubation for 1 and 3hrs in the presence or absence of 50 µg/ml of anti-C5 (eculizumab) or a mouse anti-C7 mAb, both of which blocked SBA, or by 1 to 8 µM of a factor D inhibitor (ACH-4471,Achillion) that inhibited Factor D. By ELISA, 1 µM of the factor D inhibitor completely blocked the AP at 1 µM (the lowest concentration tested) and at 8 µM (the highest concentration tested) had no effect on CP. Results. In the absence of inhibitor, blood from all five subjects incubated with bacteria showed no growth after 1 hr (&lt;50 colony forming units (cfu)/mL). For the serogroup C strain, addition of the C5 inhibitor abolished bacterial killing, as evidenced by lack of decrease in cfu/mL at 1 hr relative to time 0, and an increase of &gt;1 log10 cfu/mL at 3 hr (geometric mean, 74,200, Figure, left Panel). With the C7 inhibitor, cfu/mL decreased at 1 hr (p=0.04) and 3 hr (p=0.07) relative to time 0, and at both time points cfu/mL was significantly lower than with the anti-C5 inhibitor (p&lt;0.007). In blood from four subjects tested, addition of the Factor D inhibitor had less effect on inhibiting serogroup C bacterial killing than anti-C5 or anti-C7 as evidence by an average of 1 log10 decreased cfu/mL at 1 hr with the Factor D inhibitor relative to time 0, and &lt;50 cfu/mL at 3 hr. Similar results were obtained with the serogroup B strain by the addition of the C5 or C7 inhibitor to the blood from the three vaccinated subjects tested, and the addition of the Factor D inhibitor did not interfere with bacterial killing (&lt;50 cfu/mL at 1 hr and 3 hr, Figure, right Panel). When tests were done with blood from a serogroup B unvaccinated subject, the Factor D inhibitor blocked killing (42,000 cfu/mL at 3 hr). In experiments with blood from two vaccinated subjects and serogroup B and C strains, the addition of a C5a receptor antagonist to blood containing anti-C7 increased cfu/mL by &gt;1 log10 at 3 hr, compared to anti-C7 alone. Conclusions. Blocking C5 cleavage and release of C5a by a C5 inhibitor impairs both SBA and OPA killing of meningococci in whole blood, which provides a biologic basis for reports of failure of vaccination in preventing meningococcal disease in some patients treated witheculizumab. Blocking C7 impairs SBA but has less effect on impairing whole blood OPA killing than with anti-C5, since release of C5a, which stimulates phagocytic cell activity, is not inhibited by anti-C7. Effective killing of meningococci by blood from vaccinated subjects was seen at 3 hr (&lt; 50cfu/mL) in the presence of ACH-4471. However, ACH-4471 impaired killing of serogroup B strain by blood from an unvaccinated adult, in which bacterial killing depended on the AP. These data suggest that vaccination may be more effective in decreasing the risk of meningococcal disease in the presence of an AP inhibitor as compared to a C5 inhibitor. Figure Figure. Disclosures Granoff: Achillion Pharmaceuticals: Other: Consultant, ended May 1, 2016, Research Funding.

Lutein Leads to a Decrease of Factor D Secretion by Cultured Mature Human Adipocytes.

Purpose. Complement plays an important role in the pathogenesis of age related macular degeneration (AMD) and trials are currently being conducted to investigate the effect of complement inhibition on AMD progression. We previously found that the plasma level of factor D (FD), which is the rate limiting enzyme of the complement alternative pathway, was significantly decreased following lutein supplementation. FD is synthesized by adipose tissue, which is also the main storage site of lutein. In view of these findings we tested the hypothesis whether lutein could affect FD synthesis by adipocytes. Methods. A cell line of mature human adipocytes was incubated with 50 μg/mL lutein for 24 and 48 h, whereafter FD mRNA and protein expression were measured. Results. Lutein significantly inhibited adipocyte FD mRNA expression and FD protein release into adipocyte culture supernatants. Conclusions. Our earlier observations showing that a daily lutein supplement in individuals with early signs of AMD lowered the level of circulating FD might be caused by blocking adipocyte FD production.

Combined Inhibition of Complement and CD14 Efficiently Attenuated the Inflammatory Response Induced byStaphylococcus aureusin a Human Whole Blood Model

The complement and TLR systems are activated in sepsis, contributing to an unfavorable inflammatory "storm." Combined inhibition of these systems has been documented to efficiently attenuate the inflammatory responses induced by Gram-negative bacteria. In this study, we hypothesized that the combined inhibition would attenuate the inflammatory responses induced by Gram-positive bacteria. Staphylococcus aureus bacteria (strains Cowan and Wood), as well as S. aureus cell wall lipoteichoic acid (LTA), were incubated in thrombin-inhibited human whole blood. Complement was inhibited at the level of C3 and C5, and the TLRs by inhibiting CD14 and TLR2. Thirty-four inflammatory markers were measured by multiplex technology and flow cytometry. Thirteen markers increased significantly in response to Cowan and Wood, and 12 in response to LTA. Combined inhibition with the C3 inhibitor compstatin and the anti-CD14 Ab 18D11 significantly reduced 92 (Cowan, LTA) and 85% (Wood) of these markers. Compstatin alone significantly reduced 54 (Cowan), 38 (Wood), and 83% (LTA), whereas anti-CD14 alone significantly reduced 23, 15, and 67%, respectively. Further experiments showed that the effects of complement inhibition were mainly due to inhibition of C5a interaction with the C5a receptor. The effects on inhibiting CD14 and TLR2 were similar. The combined regimen was more efficient toward the bacterial effects than either complement or anti-CD14 inhibition alone. Complement was responsible for activation of and phagocytosis by both granulocytes and monocytes. Disrupting upstream recognition by inhibiting complement and CD14 efficiently attenuated S. aureus-induced inflammation and might be a promising treatment in both Gram-negative and Gram-positive sepsis.

Effect of complement inhibition and heparin coating on artificial surface–induced leukocyte and platelet activation

Exposure of blood to artificial surfaces, as in cardiopulmonary bypass, induces an inflammatory response involving complement, leukocyte and platelet activation. To elucidate the specific role of complement in this process, studies were performed on blood circulated in polyvinyl chloride tubing in the absence and presence of complement inhibitors. Parallel experiments were performed with heparin-coated polyvinyl chloride tubing, which is known to prevent complement and cell activation. A novel experimental model was used, based on human whole blood anticoagulated with lepirudin. Complement activation products, myeloperoxidase, lactoferrin, and thrombospondin were quantified in enzyme immunoassays. Leukocyte CD11b expression and leukocyte-platelet conjugates were detected by flow cytometry. Increased levels of C3 activation products, alternative pathway convertase, and the terminal SC5b-9 complex, combined with unchanged levels of C1rs-C1-inhibitor complexes and marginal changes in C4 activation demonstrated that complement was activated through the alternative pathway. Granulocyte and monocyte CD11b expression and granulocyte-platelet conjugate formation were efficiently attenuated by blocking either factor D, C3, C5, or C5a receptor. In contrast, monocyte-platelet conjugate formation and release of myeloperoxidase, lactoferrin, and thrombospondin were not reduced by complement inhibition. Heparin-coated polyvinyl chloride tubing efficiently reduced all inflammatory markers studied, except for C1rs-C1-inhibitor complexes, which increased, consistent with the enhancing effect of heparin on C1-inhibitor function. This effect did not, however, reduce fluid-phase classic pathway activation induced by heat-aggregated immunoglobulin G. Leukocyte and platelet activation in response to artificial materials occur by mechanisms that vary in their dependence on complement. Heparin coating precludes both the complement-dependent and complement-independent reactions.

Expression of a soluble complement inhibitor protects transgenic mice from antibody-induced acute renal failure.

Crry is a potent complement regulator in rodents that inhibits C3 convertases. In rats, intrarenal arterial injection of anti-glomerular endothelial cell (GEN) antibodies leads to complement-dependent microvascular injury and acute renal failure. In this study, a mouse variant of this model and the effects of complement inhibition were examined. Transgenic mice that overexpressed soluble Crry systemically and in their kidneys were studied. Anti-GEN IgG was injected intravenously into eight Crry transgenic mice and seven transgene-negative littermates (which were used as control animals). Thirty h after injection, blood urea nitrogen (BUN) levels were 30.3 +/- 4.4 and 114.8 +/- 23.5 mg/dl for transgene-positive and -negative animals, respectively (P = 0.012). Four of five transgene-negative animals with BUN levels of > 100 mg/dl were anuric; the remaining animal exhibited minimal albuminuria and no detectable urinary C3. In animals with renal failure, glomerular capillary collapse and tubular necrosis were observed. There was significant tubular staining for C3 in transgene-negative animals, with cellular and basal distributions, both of which were statistically greater than those in transgene-positive animals. Tubular cell C3 staining was strongly correlated with BUN values (r = 0.83, P < 0.001), as was C9 staining (r = 0.56, P = 0.037), suggesting that complement activation to the C5b-9 membrane attack complex had a casual role in renal failure. Thus, systemic injection of anti-GEN antibodies into mice leads to acute renal failure, with glomerular and tubular injury. Animals that overexpress soluble Crry in renal tubules and elsewhere are protected from the acute renal failure that occurs in this model, which ultimately seems to develop because of complement activation focused on tubules.

Hypoxic-ischemic cerebral injury in infant rats: Effect of complement inhibition

Hypoxic-ischemic cerebral injury in infant rats: Effect of complement inhibition

Aspiration-induced lung injury

To examine the role of complement in the development of acid aspiration-induced lung injury in the rat. It was postulated that inhibition or depletion of complement attenuates aspiration-induced lung injury. Controlled animal trial. Animal Laboratory, Jefferson Medical College, Philadelphia, PA. Anesthetized rats. Aspiration was induced by the intratracheal administration of 0.2 mL of 0.1 N hydrochloric acid (n = 7) and lung injury was evaluated by determining water content, myeloperoxidase activity, protein concentration, and leukocyte count in bronchoalveolar lavage fluid. Muscle PO2 was directly measured using a thin-film chamber oxygen sensor and serum tumor necrosis factor-alpha was assayed by enzyme-linked immunosorbent assay. The effect of complement inhibition by recombinant human soluble complement receptor type 1 (n = 8) or complement depletion by cobra venom factor (n = 7) on lung injury was evaluated. Acid aspiration induced pulmonary leukosequestration, edema, and a microvascular permeability defect, along with tissue hypoxia. Pretreatment with soluble complement receptor type 1 (complement inhibition) or cobra venom factor (complement depletion) significantly reduced lung edema (-61 +/- 7%; p < .05), eliminated protein accumulation in bronchoalveolar lavage fluid (p < .01), and improved (p < .05) tissue oxygenation. In contrast, there was no effect of soluble complement receptor type 1 or of cobra venom factor on leukosequestration. Acid aspiration induces lung injury through a complement-dependent mechanism that leads to microvascular permeability defects. Therefore, the possibility that complement inhibitors may have a salutary effect in humans with aspiration-induced lung injury should be investigated.

THE EFFECT OF SOLUBLE COMPLEMENT RECEPTOR TYPE 1 ON HYPERACUTE XENOGRAFT REJECTION

In the guinea pig-to-rat model of hyperacute xenograft (Xg) rejection, the effect of complement inhibition using systemically administered soluble complement receptor type 1 (sCR1) on discordant cardiac Xg survival was investigated. In PBS-treated control Xg recipients (n = 13), hyperacute rejection was rapid, with a mean Xg survival of 17 +/- 4 min. Therapy with sCR1 prolonged survival of cardiac Xgs in a dose-dependent manner. A 3 mg/kg bolus of sCR1 (n = 4) prolonged Xg survival to 64 +/- 29 min (not significant). Increasing the sCR1 dose to 5.9 mg/kg (n = 4) significantly delayed Xg rejection to 71 +/- 17 min (P-0.026, log-rank test vs. control). In 10 recipients treated with 15 mg/kg sCR1, mean Xg survival was further prolonged to 189 +/- 36 min (P-0.0004) with no adverse effects. While 2 of 8 recipients receiving 60 mg/kg sCR1 died with functioning Xgs at 30 and 300 min due to anastomotic bleeding, Xg survival averaged over 12 hr (747 +/- 100 min, P-0.0004) in the remaining 6 recipients. sCR1 administration significantly inhibited serum complement activity in a parallel dose-dependent fashion, with the 60 mg/kg dose reducing complement activity by 95 +/- 1 and 96 +/- 1% five and 30 min following Xg reperfusion, respectively. Immunofluorescence microscopy revealed rat IgM bound to all cardiac Xgs in control as well as sCR1-treated recipients. In addition, serial histologic examination of cardiac Xgs harvested within 21 min of graft reperfusion revealed occlusive platelet aggregates within the coronary vessels as well as interstitial hemorrhage and myocardial necrosis in Xgs from control recipients, all of which were only minimally present in Xgs from recipients treated with sCR1. These studies show that complement inhibition with sCR1 significantly delays hyperacute cardiac Xg rejection in this discordant model and may be an important component in a therapeutic protocol for xenotransplantation.