Endogenous Complement Research Articles

Endogenous complement 1q binding protein (C1qbp) regulates mitochondrial permeability transition and post-myocardial infarction remodeling and dysfunction

The mitochondrial permeability transition (MPT) pore regulates necrotic cell death following diverse cardiac insults. While the componentry of the pore itself remains controversial, Cyclophilin D (CypD) has been well-established as a positive regulator of pore opening. We have previously identified Complement 1q-binding protein (C1qbp) as a novel CypD-interacting molecule and a negative regulator of MPT-dependent cell death in vitro. However, its effects on the MPT pore and sensitivity to cell death in the heart remain untested. We therefore hypothesized that C1qbp would inhibit MPT in cardiac mitochondria and protect cardiac myocytes against cell death in vivo. To investigate the effects of C1qbp in the myocardium we generated gain- and loss-of-function mice. Transgenic C1qbp overexpression resulted in decreased complex protein expression and reduced mitochondrial respiration and ATP production but MPT was unaffected. In contrast, while C1qbp+/− mice did not exhibit any changes in mitochondrial protein expression, respiration, or ATP, the MPT pore was markedly sensitized to Ca2+ in these animals. Neither overexpression nor depletion of C1qbp significantly affected baseline heart morphology or function at 3 months of age. When subjected to myocardial infarction, C1qbp transgenic mice exhibited similar infarct sizes and cardiac remodeling to non-transgenic mice, consistent with the lack of an effect on MPT. In contrast, cardiac scar formation and dysfunction were significantly increased in the C1qbp+/− mice compared to C1qbp+/+ controls. Our results suggest that C1qbp is required for normal regulation of the MPT pore and mitochondrial function, and influences cardiac remodeling following MI, the latter more likely being independent of C1qbp effects on the MPT pore.

Targeted complement inhibition using bispecific antibodies that bind local antigens and endogenous complement regulators.

Complement activation protects against infection but also contributes to pathological mechanisms in a range of clinical conditions such as autoimmune diseases and transplant rejection. Complement-inhibitory drugs, either approved or in development, usually act systemically, thereby increasing the risk for infections. We therefore envisioned a novel class of bispecific antibodies (bsAbs) which are capable of site-directed complement inhibition by bringing endogenous complement regulators in the vicinity of defined cell surface antigens. Here, we analyzed a comprehensive set of obligate bsAbs designed to crosslink a specific target with either complement regulator factor H (FH) or C4b-binding protein (C4BP). The bsAbs were assessed for their capacity to inhibit complement activation and cell lysis in an antigen-targeted manner. We observed that the bsAbs inhibited classical, lectin, and alternative pathway complement activation in which sufficient endogenous serum FH and C4BP could be recruited to achieve local inhibition. Importantly, the bsAbs effectively protected antigen-positive liposomes, erythrocytes, and human leukocytes from complement-mediated lysis. In conclusion, localized complement inhibition by bsAbs capable of recruiting endogenous human complement regulators (such as FH or C4BP) to cell surfaces potentially provides a novel therapeutic approach for the targeted treatment of complement-mediated diseases.

The interactive effect of endogenous and exogenous institutions on forest use practices in socio-culturally diverse landscapes of Cameroon

AbstractIn resource-dependent communities of sub-Saharan Africa, it is widely held that endogenous institutions either countervail or complement exogenous institutions, indicating inconsistency in empirical evidence. This has re-ignited interest to establish the independent and/or interactive effect of both endogenous and exogenous institutional structures. This paper quantitatively analyses the independent and interactive effects of community-based (endogenous) and state-based (exogenous) forest management institutions (FMIs) on the exploitation of forest resources, drawing from a representative sample of 446 households from three socio-culturally diverse landscapes of Cameroon. The paper introduces a multiplicative interaction term and used the heteroscedasticity-based instrumental variables approach to estimate the direct and indirect effects of exogenous and endogenous institutions on the behaviour of users of forest-based resources. The results indicate that: (i) Both exogenous structures of FMIs and the interaction term significantly constrain the extractive behaviour of forest resource users; (ii) endogenous structures and not rules are vital in improving the role of exogenous structures in forest resource management; (iii) while traditional rules are less significant in shaping forest resource exploitation, their effectiveness is highly significant across the study sites and (iv) institutional compatibility and complementarity was observed in landscapes that were formerly shaped by British colonial influence (Bakossi and Kilum-Ijim). This paper does not only establish the relative influence of exogenous and endogenous FMIs, it also highlights future research pathways on institutional change in the context of environmental resource management in sub-Saharan Africa.

99 Targeted complement inhibition using engineered bispecific antibodies that bind local antigens and endogenous complement regulators as a novel therapeutic approach

99 Targeted complement inhibition using engineered bispecific antibodies that bind local antigens and endogenous complement regulators as a novel therapeutic approach

Abstract 1312: Pro-survival lipid metabolism activates intracellular complement signaling to induce inflammasome-mediated tumor metastasis

Abstract Tumor metastasis remains one of the major causes of cancer-related deaths in patients with solid tumors. Mechanisms by which metabolic alterations regarding pro-survival lipid signaling activation in inducing cancer cell migration and metastasis are largely unknown. Here, we demonstrate that sphingosine 1-phosphate (S1P) metabolism activates endogenous complement signaling for inducing tumor metastasis via inducing inflammasome-mediated pro-inflammation. Our studies using molecular, pharmacologic, and genetic tools showed that activation of S1P and S1P receptor 1 (S1P/S1PR1) induces tumor metastasis via enhanced intracellular complement signaling by the regulation of the C3-PPIL1 complex and subsequent inflammasome activation in cancer cells and in vivo xenograft-derived tumors. Citation Format: Alhaji H. Janneh, Besim Ogretmen. Pro-survival lipid metabolism activates intracellular complement signaling to induce inflammasome-mediated tumor metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1312.

1467-P: Chemical Profiles of Acinar Tissue and Pancreatic Islets from Healthy and T2D-Affected Pancreata

Both the endocrine and exocrine pancreas demonstrate morphological and chemical changes associated with T2D development. Despite significant progress, chemical profiles of the islets and acinar tissue following disease progression are not well characterized. In this work we investigated isolated populations of pancreatic islets (n=18) and acinar tissues (n=7) collected from healthy and T2D affected pancreata using several mass spectrometry (MS) approaches. Samples are provided by the ADI IsletCore and the Network for Pancreatic Organ Donors with Diabetes (nPOD) . Both peptide and metabolite profiles were determined and compared via chemical profiling with matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance MS. Not surprisingly, the peptide profiles from T2D islets demonstrated lower signal intensities compared to healthy islets (ROC analysis, AUC=0.9) . Somatostatin, pancreatic icosapeptide, and insulin-related signals are most decreased. Preliminary data for acinar tissues indicate lower peptide levels in samples collected from T2D-affected (n=2) vs. healthy (n=5) pancreata. Several unique peptides are detected specifically in acinar tissue obtained from T2D-affected pancreata. Liquid chromatography-MS reveals a complex endogenous peptide complement of acinar tissue. Analysis of extracellular media for both islet types demonstrated similar differences (AUC=0.7) indicating higher levels of observed peptides in preparations from healthy islets, perhaps suggesting that peptides are released from the islets during shipment. Integrity of received islets was verified using dithizone staining prior to measurement. In contrast, almost no peptide markers of acinar tissue are found in corresponding extracellular media. Our results enhance knowledge on biochemical signature of T2D in the pancreas. Disclosure S.Rubakhin: None. E.V.Romanova: None. J.V.Sweedler: None. Funding American Diabetes Association (1-18-VSN-19)

Depletion of Toxoplasma adenine nucleotide translocator leads to defects in mitochondrial morphology

BackgroundAdenine nucleotide translocase (ANT) is a protein that catalyzes the exchange of ADP/ATP across the inner mitochondrial membrane. Beyond this, ANT is closely associated with cell death pathways and mitochondrial dysfunction. It is a potential therapeutic target for many diseases. The function of the ANT in Toxoplasma gondii is poorly understood.MethodsThe CRISPR/CAS9 gene editing tool was used to identify and study the function of the ANT protein in T. gondii. We constructed T. gondii ANT transgenic parasite lines, including endogenous tag strain, knockout strain and gene complement strain, to clarify the function and location of TgANT. Mitochondrial morphology was observed by immunofluorescence and transmission electron microscopy.ResultsToxoplasma gondii was found to encode an ANT protein, which was designated TgANT. TgANT localized to the inner mitochondrial membrane. The proliferation of the Δant strain was significantly reduced. More important, depletion of TgANT resulted in significant changes in the morphology and ultrastructure of mitochondria, abnormal apicoplast division and abnormal cytoskeletal daughter budding. In addition, the pathogenicity of the Δant strain to mice was significantly reduced.ConclusionsAltogether, we identified and characterized the ANT protein of T. gondii. Depletion of TgANT inhibited parasite growth and impaired apicoplast and mitochondrial biogenesis, as well as abnormal parasite division, suggesting TgANT is important for parasite growth.

Complement C3 deficiency enhances anti-CD47 efficacy in murine ovarian cancer model.

Abstract Epithelial ovarian cancer (OC) is the most lethal gynecological malignancy. While OC is an immunogenic tumor, single-agent anti-PD-1 have limited efficacy in recurrent OC. Blockade of the CD47/SIRPα pathway, a phagocytosis checkpoint in macrophages and other myeloid cells, can enhance myeloid-driven antitumor immunity and tumor control. However, the benefit of targeting CD47/SIRPα in recurrent OC has been limited. Prior work from our lab and others point to complement priming of innate immune responses as a barrier to T-cell-driven anti-tumor immunity. However, in the specific context of anti-CD47 therapy, complement opsonization of tumor cells could augment the capacity of myeloid cells to phagocytose or injure tumor cells. The role of complement deficiency with anti-CD47 therapy in tumor control is a gap in the field, we therefore evaluated the efficacy of anti-CD47 in female wild-type (WT) versus C3−/− and C5aR1−/− with syngeneic OC (ID8). Mice (n=8–10 per group) were administered IP ID8 tumor cells followed by anti-CD47 or isotype twice weekly for 3 weeks and were monitored for the ascites development and survival. On day 84, all WT mice treated with isotype had ascites by visual inspection and 3 of 10 died. The proportion of mice with ascites varied in other groups, C3−/− mice treated with anti-CD47 having the lowest proportion, 4 of 9 mice with ascites and no deaths (Fisher’s test, WT-isotype vs C3−/− + anti-CD47, p= 0.01). A limitation is the potential endogenous complement production by tumor cells, which will be evaluated. These results suggest a potential benefit for dual targeting of the CD47/SIRPα and complement pathways in OC. Additional studies will probe immunologic correlates and survival benefits of these therapies in OC. This work was supported by Roswell Park Cancer Center Support Grant P30CA016056 and Apellis Pharmaceuticals, Inc

Pro‐survival lipid metabolism activates intracellular complement signaling to induce inflammasome‐mediated tumor metastasis

Tumor metastasis remains one of the major causes of cancer‐related deaths in patients with solid tumors. Mechanisms by which metabolic alterations regarding pro‐survival lipid signaling activation in inducing cancer cell migration and metastasis are largely unknown. Here, we demonstrate that sphingosine 1‐phosphate (S1P) metabolism activates endogenous complement signaling for inducing tumor metastasis via inducing inflammasome‐mediated pro‐inflammation. Our studies using molecular, pharmacologic, and genetic tools showed that activation of S1P and S1P receptor 1 (S1P/S1PR1) induces tumor metastasis via enhanced intracellular complement signaling by the regulation of the C3‐PPIL1 complex and subsequent inflammasome activation in cancer cells and in vivo xenograft‐derived tumors.

The Complement Pathway: New Insights into Immunometabolic Signaling in Diabetic Kidney Disease.

Significance:The metabolic disorder, diabetes mellitus, results in microvascular complications, including diabetic kidney disease (DKD), which is partly believe to involve disrupted energy generation in the kidney, leading to injury that is characterized by inflammation and fibrosis. An increasing body of evidence indicates that the innate immune complement system is involved in the pathogenesis of DKD; however, the precise mechanisms remain unclear.Recent Advances:Complement, traditionally thought of as the prime line of defense against microbial intrusion, has recently been recognized to regulate immunometabolism. Studies have shown that the complement activation products, Complement C5a and C3a, which are potent pro-inflammatory mediators, can mediate an array of metabolic responses in the kidney in the diabetic setting, including altered fuel utilization, disrupted mitochondrial respiratory function, and reactive oxygen species generation. In diabetes, the lectin pathway is activated via autoreactivity toward altered self-surfaces known as danger-associated molecular patterns, or via sensing altered carbohydrate and acetylation signatures. In addition, endogenous complement inhibitors can be glycated, whereas diet-derived glycated proteins can themselves promote complement activation, worsening DKD, and lending support for environmental influences as an additional avenue for propagating complement-induced inflammation and kidney injury.Critical Issues:Recent evidence indicates that conventional renoprotective agents used in DKD do not target the complement, leaving this web of inflammatory stimuli intact.Future Directions:Future studies should focus on the development of novel pharmacological agents that target the complement pathway to alleviate inflammation, oxidative stress, and kidney fibrosis, thereby reducing the burden of microvascular diseases in diabetes. Antioxid. Redox Signal. 37, 781–801.

Status of mannose-binding lectin (MBL) and complement system in COVID-19 patients and therapeutic applications of antiviral plant MBLs.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by a virus called “Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).” In the majority of patients, infection with COVID-19 may be asymptomatic or may cause only mild symptoms. However, in some patients, there can also be immunological problems, such as macrophage activation syndrome (CSS) that results in cytokine storm syndrome (CSS) and acute respiratory distress syndrome (ARDS). Comprehension of host-microbe communications is the critical aspect in the advancement of new therapeutics against infectious illnesses. Endogenous animal lectins, a class of proteins, may perceive non-self glycans found on microorganisms. Serum mannose-binding lectin (sMBL), as a part of the innate immune framework, recognizes a wide range of microbial microorganisms and activates complement cascade via an antibody-independent pathway. Although the molecular basis for the intensity of SARS-CoV-2 infection is not generally understood, scientific literature indicates that COVID-19 is correlated with unregulated activation of the complement in terms of disease severity. Disseminated intravascular coagulation (DIC), inflammation, and immune paralysis contribute to unregulated complement activation. Pre-existing genetic defects in MBL and their association with complement play a major role in immune response dysregulation caused by SARS-CoV-2. In order to generate anti-complement-based therapies in Covid-19, an understanding of sMBL in immune response to SARS-CoV-2 and complement is therefore essential. This review highlights the role of endogenous sMBL and complement activation during SARS-CoV-2 infection and their therapeutic management by various agents, mainly plant lectins, since antiviral mannose-binding plant lectins (pMBLs) offer potential applications in the prevention and control of viral infections.

An Immunoregulatory Role for Complement Receptors in Murine Models of Breast Cancer.

The complement system has demonstrated roles in regulating tumor growth, although these may differ between tumor types. The current study used two murine breast cancer models (EMT6 and 4T1) to investigate whether pharmacological targeting of receptors for complement proteins C3a (C3aR) and C5a (C5aR1) is protective in murine breast cancer models. In contrast to prior studies in other tumor models, treatment with the selective C5aR1 antagonist PMX53 had no effect on tumor growth. However, treatment of mice with a dual C3aR/C5aR1 agonist (YSFKPMPLaR) significantly slowed mammary tumor development and progression. Examination of receptor expression by quantitative polymerase chain reaction (qPCR) analysis showed very low levels of mRNA expression for either C3aR or C5aR1 by EMT6 or 4T1 mammary carcinoma cell lines compared with the J774 macrophage line or bone marrow-derived macrophages. Moreover, flow cytometric analysis found no evidence of C3aR or C5aR1 protein expression by either EMT6 or 4T1 cells, leading us to hypothesize that the tumor inhibitory effects of the dual agonist are indirect, possibly via regulation of the anti-tumor immune response. This hypothesis was supported by flow cytometric analysis of tumor infiltrating leukocyte populations, which demonstrated a significant increase in T lymphocytes in mice treated with the C3aR/C5aR1 agonist. These results support an immunoregulatory role for complement receptors in primary murine mammary carcinoma models. They also suggest that complement activation peptides can influence the anti-tumor response in different ways depending on the cancer type, the host immune response to the tumor and levels of endogenous complement activation within the tumor microenvironment.

Crovalimab for Treatment of Patients with Paroxysmal Nocturnal Hemoglobinuria and Complement C5 Polymorphism - Experience from the Phase I/II COMPOSER Study

Crovalimab for Treatment of Patients with Paroxysmal Nocturnal Hemoglobinuria and Complement C5 Polymorphism - Experience from the Phase I/II COMPOSER Study

The endogenous neuronal complement inhibitor SRPX2 protects against complement-mediated synapse elimination during development.

Complement-mediated synapse elimination has emerged as an important process in both brain development and neurological diseases, but whether neurons express complement inhibitors that protect synapses against complement-mediated synapse elimination remains unknown. Here, we show that the sushi domain protein SRPX2 is a neuronally expressed complement inhibitor that regulates complement-dependent synapse elimination. SRPX2 directly binds to C1q and blocks its activity, and SRPX2−/Y mice show increased C3 deposition and microglial synapse engulfment. They also show a transient decrease in synapse numbers and increase in retinogeniculate axon segregation in the lateral geniculate nucleus. In the somatosensory cortex, SRPX2−/Y mice show decreased thalamocortical synapse numbers and increased spine pruning. Dual C3−/−;SRPX2−/Y mice exhibit phenotypes associated with C3−/− mice rather than SRPX2−/Y mice, indicating that C3 is necessary for SRPX2’s effect on synapse elimination. Together, these results show that SRPX2 protects synapses against complement-mediated elimination in both the thalamus and the cortex.

Microglia complement astrocytes in neuromyelitis optica.

Neuromyelitis optica (NMO) is a central nervous system (CNS) inflammatory autoimmune disease caused by antibodies against aquaporin-4 (AQP4) expressed on astrocytes. Binding of AQP4-specific antibodies (NMO-IgG) triggers activation of the complement cascade, which is responsible for astrocyte loss and secondary demyelination. Although the role for the cytolytic complement proteins in astrocyte destruction in NMO is well established, little is known regarding the initial phase of astrocyte injury. In this issue of the JCI, Chen and colleagues evaluated the precytolytic phase when NMO-IgG binds astrocytes in vivo in the absence of exogenous complement. NMO-IgG alone caused astrocyte activation and AQP4 loss. Surprisingly, microglia, CNS-resident innate immune cells that produce endogenous complement, were required for clinical manifestations of disease, a finding that suggests microglia may serve as a therapeutic target in NMO.

Expression and production of the SERPING1-encoded endogenous complement regulator C1-inhibitor in multiple cohorts of tuberculosis patients

BackgroundTo facilitate better discrimination between patients with active tuberculosis (TB) and latent TB infection (LTBI), whole blood transcriptomic studies have been performed to identify novel candidate host biomarkers. SERPING1, which encodes C1-inhibitor (C1-INH), the natural inhibitor of the C1-complex has emerged as candidate biomarker. Here we collated and analysed SERPING1 expression data and subsequently determined C1-INH protein levels in four cohorts of patients with TB. MethodsSERPING1 expression data were extracted from online deposited datasets. C1-INH protein levels were determined by ELISA in sera from individuals with active TB, LTBI as well as other disease controls in geographically diverse cohorts. FindingsSERPING1 expression was increased in patients with active TB compared to healthy controls (8/11 cohorts), LTBI (13/14 cohorts) and patients with other (non-TB) lung-diseases (7/7 cohorts). Serum levels of C1-INH were significantly increased in The Gambia and Italy in patients with active TB relative to the endemic controls but not in South Africa or Korea. In the largest cohort (n = 50), with samples collected longitudinally, normalization of C1-INH levels following successful TB treatment was observed. This cohort, also showed the most abundant increase in C1-INH, and a positive correlation between C1q and C1-INH levels. Combined presence of increased levels of both C1q and C1-INH had high specificity for active TB (96 %) but only very modest sensitivity 38 % compared to the endemic controls. InterpretationSERPING1 transcript expression is increased in TB patients, while serum protein levels of C1-INH were increased in half of the cohorts analysed.

Oxidative Stress Increases Endogenous Complement-Dependent Inflammatory and Angiogenic Responses in Retinal Pigment Epithelial Cells Independently of Exogenous Complement Sources.

Oxidative stress-induced damage of the retinal pigment epithelium (RPE) and chronic inflammation have been suggested as major contributors to a range of retinal diseases. Here, we examined the effects of oxidative stress on endogenous complement components and proinflammatory and angiogenic responses in RPE cells. ARPE-19 cells exposed for 1–48 h to H2O2 had reduced cell–cell contact and increased markers for epithelial–mesenchymal transition but showed insignificant cell death. Stressed ARPE-19 cells increased the expression of complement receptors CR3 (subunit CD11b) and C5aR1. CD11b was colocalized with cell-derived complement protein C3, which was present in its activated form in ARPE-19 cells. C3, as well as its regulators complement factor H (CFH) and properdin, accumulated in the ARPE-19 cells after oxidative stress independently of external complement sources. This cell-associated complement accumulation was accompanied by increased nlrp3 and foxp3 expression and the subsequently enhanced secretion of proinflammatory and proangiogenic factors. The complement-associated ARPE-19 reaction to oxidative stress, which was independent of exogenous complement sources, was further augmented by the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib. Our results indicate that ARPE-19 cell-derived complement proteins and receptors are involved in ARPE-19 cell homeostasis following oxidative stress and should be considered as targets for treatment development for retinal degeneration.

Interactions between the complement and endothelin systems in normal pregnancy and following placental ischemia

Preeclampsia is characterized by new onset hypertension and fetal growth restriction and is associated with aberrant activation of the innate immune complement system and stressed or ischemic placenta. Previous studies have suggested a role for both endothelin and complement system activation products in new onset hypertension in pregnancy, but inter-relationships of the pathways are unclear. We hypothesized that complement activation following placental ischemia stimulates the endothelin pathway to cause hypertension and impair fetal growth. The Reduced Uterine Perfusion Pressure (RUPP) model results in hypertension and fetal growth restriction in a pregnant rat due to placental ischemia caused by mechanical obstruction of blood flow to uterus and placenta. The effect of inhibitor of complement activation soluble Complement Receptor 1 (sCR1) and endothelin A receptor (ETA) antagonist atrasentan on hypertension, fetal weight, complement activation (systemic circulating C3a and local C3 placental deposition) and endothelin [circulating endothelin and message for preproendothelin (PPE), ETA and endothelin B receptor (ETB) in placenta] in the RUPP rat model were determined. Following placental ischemia, sCR1 attenuated hypertension but increased message for PPE and ETA in placenta, suggesting complement activation causes hypertension via an endothelin independent pathway. With ETA antagonism the placental ischemia-induced increase in circulating C3a was unaffected despite inhibition of hypertension, indicating systemic C3a alone is not sufficient. In normal pregnancy, inhibiting complement activation increased plasma endothelin but not placental PPE message. Atrasentan treatment increased fetal weight, circulating endothelin and placental ETA message, and unexpectedly increased local complement activation in placenta (C3 deposition) but not C3a in circulation, suggesting endothelin controls local placental complement activation in normal pregnancy. Atrasentan also significantly decreased message for endogenous complement regulators Crry and CD55 in placenta and kidney in normal pregnancy. Results of our study indicate that complement/endothelin interactions differ in pregnancies complicated with placental ischemia vs normal pregnancy, as well as locally vs systemically. These data clearly illustrate the complex interplay between complement and endothelin indicating that perturbations of either pathway may affect pregnancy outcomes.

Exploring the structural similarity yet functional distinction between coagulation factor XIII-B and complement factor H sushi domains.

Coagulation factor XIII (FXIII) covalently crosslinks pre-formed fibrin clots preventing their premature fibrinolysis. In plasma, FXIII circulates as a zymogenic heterotetramer composed of catalytic FXIII-A subunits, and carrier/regulatory FXIII-B subunits. FXIII-A is a well characterized component of this complex, and has been associated with several pleiotropic roles outside coagulation as well. In comparison only protective/regulatory roles towards the FXIII-A subunit have been identified for FXIII-B. Strong homology between FXIII-B and complement regulator Complement factor H suggests a putative role of FXIII-B in complement activation. In the current study we have analyzed the similarities and yet functional divergence of these two proteins using in silico sequence alignment and structural analysis. We have evaluated complement activation post reconstitution of FXIII components into FXIII deficient and CFH deficient plasma. We have also transiently expressed FXIII-B in SH-SY5Y cell lines and evaluated its effect on the endogenous complement activation. Our investigations show no effect of FXIII-B subunit on the rate of complement activation. Therefore we conclude that at a physiological level, FXIII-B subunit plays no role in the complement system, although a vestigial function in altered pathological states might still exist.

The Importance of Eculizumab in Paroxysmal Nocturnal Hemoglobinuria: A Cohort Study

IntroductionParoxysmal nocturnal hemoglobinuria (PNH) is a rare, often progressively debilitating and life-threatening acquired disease characterized by chronic, complement-mediated intravascular hemolysis. PNH is characterized by the deficiency of the endogenous glycosyl phosphatidylinositol-anchored complement regulatory proteins CD55 and CD59 and other proteins on the surface of blood cells, which results in chronic uncontrolled activation of the complement system at the site of the PNH cells and intravascular hemolysis and thrombosis. PNH may present in the absence of an underlying bone marrow disorder (BMD), as a condition secondary to BMDs, such as aplastic anemia (AA) or myelodysplastic syndrome, or as sub-clinical PNH. In this cohort study we will report clinical findings, management options and eculizumab response of 38 PNH cases in our clinic.Materials and MetodWe followed 38 PNH cases in this cohort study. We used flow cytometric PNH clone detection test since 1993 but HAM's test and sucrose lysis test were the only tests for the PNH diagnosis in older cases. We used eculizumab in 20 of our patients who had transfusion dependent chronic intravascular hemolysis and/or thrombosis. All of of the patients were vaccinated for Neisseria meningitides, Pneumococcus and Hemophilus influenza type B prior to 2 weeks of initiation of Eculizumab. Eculizumab was administered 600 mg/day for 4 weeks and dosage was elevated to 900 mg/day at week 5, then 900 mg/day every other week as maintenance therapy. The response to therapy was evaluated by LDH, blood cell counts and haptoglobin levels. Clinical and laboratory findings, management options and treatment effect on survival curves were evaluated.FindingsThirty-eight PNH patients who were followed in our clinic since 1985 were enrolled in this study. Twenty of 38 were female and the median age was 35 years (ranging from 18 - 62). The oldest one has been followed for 32 years. Chronic hemolytic anemia and at least one lineage of cytopenia were seen in each patient during diagnosis.Fifteen patients were diagnosed as AA and were treated with immunosuppressive therapy before PNH diagnosis. Following AA diagnosis, PNH was diagnosed on average 35 months (three to 192 months) . One of the patients was diagnosed as MDS and followed-up for five years with this diagnosis. Twenty-two of the cases were de-novo PNH disease. Thrombotic complications were seen at the diagnosis in two patients. Unfortunately, we also have seen thrombotic complications in 10 more patients during follow-up period before eculizumab availability.Five of our patients were lost on eight, 14, 60, 86 and 260 months after their diagnosis. We lost two of them with infection and two of them with thrombotic complications. Fifth patient that we lost at 260 months of her follow-up had complete response with eculizumab and was deceased following a fall from height. Two of our patients went to abroad for treatment. Six of them were lost to follow-up. We currently follow 25 of 38 patients. We still use eculizumab in 20 of our patients. We used eculizumab in median 8 months after PNH diagnosis (One to 360 months). In 15 patients, transfusion dependent chronic intravascular hemolysis was the main indication and for the remaining 5 patients thrombosis was the main indication. Quality of life parameters changed significantly in all of the patients and we didn't observe any new thrombotic episodes. We used eculizumab as an important treatment option for a median 63 months (eight to 98 months) and we didn't encounter any side effects other than mild headache and flu-like symptoms in 5 of the cases.Discussion and conclusionPNH is a life-threatening disease and four of the patients in our cohort died in median 37 months of their diagnosis. Although thrombosis was the main mortality and morbidity reason in our cohort, we didn't see any thrombotic episodes under eculizumab treatment. Even in induction period of the therapy, both clinical and laboratory findings improved significantly with Eculizumab. None of our patients needed transfusion on eculizumab treatment. The survival of our PNH patients on eculizumab is excellent and we didn't lose any of our patients since induction of eculizumab therapy.In conclusion, eculizumab is the first disease-modifying, targeted therapy for PNH and its availability has radically changed the management of patients with this rare disease. DisclosuresYenerel:Alexion: Honoraria, Speakers Bureau.