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In adenovirus type 5 (HAdV-5)-derived viral vectors, the fiber protein has been the preferred locale for modifications to alter the natural viral tropism. Hexon, the most abundant capsid protein, has rarely been used for retargeting purposes, likely because the insertion of larger targeting peptides into Hexon often interferes with the assembly of the viral capsid. We previously observed that positively charged molecules enhance the transduction of human multipotent mesenchymal stromal cells (hMSCs)-a cell type of significant interest for clinical development but inefficiently transduced by unmodified HAdV-5-based vectors. As efficient HAdV-5-mediated gene transfer would greatly increase the therapeutic potential of hMSCs, we tested the hypothesis that introducing positively charged amino acids into Hexon might enhance the transduction of hMSCs, enabling efficient expression of selected transgenes. From the constructs that could be rescued as functional virions, one (HAdV-5-HexPos3) showed striking transduction of hMSCs with up to 500-fold increased efficiency. Evaluation of the underlying mechanism identified heparan sulfate proteoglycans (HSPGs) to be essential for virus uptake by the cells. The ease and efficiency of transduction of hMSCs with this vector will facilitate the development of genetically modified hMSCs as therapeutic vehicles in different disciplines, including oncology or regenerative medicine.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hemolytic disease driven by impaired complement regulation. Mutations in genes encoding the enzymes that build the GPI anchors are causative, with somatic mutations in the PIG-A gene occurring most frequently. As a result, the important membrane-bound complement regulators CD55 and CD59 are missing on the affected hematopoietic stem cells and their progeny, rendering those cells vulnerable to complement attack. Immune escape mechanisms sparing affected PNH stem cells from removal are suspected in the PNH pathogenesis, but molecular mechanisms have not been elucidated. We hypothesized that exuberant complement activity in PNH results in enhanced immune checkpoint interactions, providing a molecular basis for the potential immune escape in PNH. In a series of PNH patients, we found increased expression levels of the checkpoint ligand programmed death-ligand 1 (PD-L1) on granulocytes and monocytes, as well as in the plasma of PNH patients. Mechanistically, we demonstrate that complement activation leading to the decoration of particles/cells with C3- and/or C4-opsonins increased PD-L1 expression on neutrophils and monocytes as shown for different in vitro models of classical or alternative pathway activation. We further establish in vitro that complement inhibition at the level of C3, but not C5, inhibits the alternative pathway–mediated upregulation of PD-L1 and show by means of soluble PD-L1 that this observation translates into the clinical situation when PNH patients are treated with either C3 or C5 inhibitors. Together, the presented data show that the checkpoint ligand PD-L1 is increased in PNH patients, which correlates with proximal complement activation.

The use of biomaterials in modern medicine has enabled advanced drug delivery strategies and led to reduced morbidity and mortality in a variety of interventions such as transplantation or hemodialysis. However, immune-mediated reactions still present a serious complication of these applications. One of the drivers of such reactions is the complement system, a central part of humoral innate immunity that acts as a first-in-line defense system in its own right but also coordinates other host defense responses. A major regulator of the complement system is the abundant plasma protein factor H (FH), which impairs the amplification of complement responses. Previously, we could show that it is possible to recruit FH to biomedical surfaces using the phage display-derived cyclic peptide 5C6 and, consequently, reduce deposition of C3b, an activation product of the complement system. However, the optimal orientation of 5C6 on surfaces, structural determinants within the peptide for the binding, and the exact binding region on FH remained unknown. Here, we show that the cyclic core and C-terminal region of 5C6 are essential for its interaction with FH and that coating through its N-terminus strongly increases FH recruitment and reduces C3-mediated opsonization in a microparticle-based assay. Furthermore, we could demonstrate that 5C6 selectively binds to FH but not to related proteins. The observation that 5C6 also binds murine FH raises the potential for translational evaluation in animal models. This work provides important insight for the future development of 5C6 as a probe or therapeutic entity to reduce complement activation on biomaterials. STATEMENT OF SIGNIFICANCE: Biomaterials have evolved into core technologies critical to biomedical and drug delivery applications alike, yet their safe and efficient use may be adversely impacted by immune responses to the foreign materials. Taking inspiration from microbial immune evasion strategies, our group developed a peptide-based surface coating that recruits factor H (FH), a host regulator of the complement system, from plasma to the material surface and prevents unwanted activation of this innate immunity pathway. In this study, we identified the molecular determinants that define the interaction between FH and the coated peptide, developed tethering strategies with largely enhanced binding capacity and provided important insight into the target selectivity and species specificity of the FH-binding peptide, thereby paving the way for preclinical development steps.

Blocking the terminal complement pathway with the C5 inhibitor eculizumab has revolutionized the clinical management of several complement-mediated diseases and has boosted the clinical development of new inhibitors. Data on the C3 inhibitor Compstatin and the C5 inhibitors eculizumab and Coversin reported here demonstrate that C3/C5 convertases function differently from prevailing concepts. Stoichiometric C3 inhibition failed to inhibit C5 activation and lytic activity during strong classical pathway activation, demonstrating a “C3 bypass” activation of C5. We show that, instead of C3b, surface-deposited C4b alone can also recruit and prime C5 for consecutive proteolytic activation. Surface-bound C3b and C4b possess similar affinities for C5. By demonstrating that the fluid phase convertase C3bBb is sufficient to cleave C5 as long as C5 is bound on C3b/C4b-decorated surfaces, we show that surface fixation is necessary only for the C3b/C4b opsonins that prime C5 but not for the catalytic convertase unit C3bBb. Of note, at very high C3b densities, we observed membrane attack complex formation in absence of C5-activating enzymes. This is explained by a conformational activation in which C5 adopts a C5b-like conformation when bound to densely C3b-opsonized surfaces. Stoichiometric C5 inhibitors failed to prevent conformational C5 activation, which explains the clinical phenomenon of residual C5 activity documented for different inhibitors of C5. The new insights into the mechanism of C3/C5 convertases provided here have important implications for the development and therapeutic use of complement inhibitors as well as the interpretation of former clinical and preclinical data.

Rising incidences and mortalities have drawn attention to

Over a century after the discovery of the complement system, the first complement therapeutic was approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH). It was a long-acting monoclonal antibody (aka 5G1-1, 5G1.1, h5G1.1, and now known as eculizumab) that targets C5, specifically preventing the generation of C5a, a potent anaphylatoxin, and C5b, the first step in the eventual formation of membrane attack complex. The enormous clinical and financial success of eculizumab across four diseases (PNH, atypical hemolytic uremic syndrome (aHUS), myasthenia gravis (MG), and anti-aquaporin-4 (AQP4) antibody-positive neuromyelitis optica spectrum disorder (NMOSD)) has fueled a surge in complement therapeutics, especially targeting diseases with an underlying complement pathophysiology for which anti-C5 therapy is ineffective. Intensive research has also uncovered challenges that arise from C5 blockade. For example, PNH patients can still face extravascular hemolysis or pharmacodynamic breakthrough of complement suppression during complement-amplifying conditions. These “side” effects of a stoichiometric inhibitor like eculizumab were unexpected and are incompatible with some of our accepted knowledge of the complement cascade. And they are not unique to C5 inhibition. Indeed, “exceptions” to the rules of complement biology abound and have led to unprecedented and surprising insights. In this review, we will describe initial, present and future aspects of protein inhibitors of the complement cascade, highlighting unexpected findings that are redefining some of the mechanistic foundations upon which the complement cascade is organized., publishedVersion

In 2007 and 2009, the regulatory approval of the first-in-class complement inhibitor eculizumab revolutionized the clinical management of 2 rare, life-threatening clinical conditions: paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). Although being completely distinct diseases affecting blood cells and the glomerulus, PNH and aHUS remarkably share several features in their etiology and clinical presentation. An imbalance between complement activation and regulation at host surfaces underlies both diseases precipitating in severe thrombotic events that are largely resistant to anticoagulant and/or antiplatelet therapies. Inhibition of the common terminal complement pathway by eculizumab prevents the frequently occurring thrombotic events responsible for the high mortality and morbidity observed in patients not treated with anticomplement therapy. Although many in vitro and ex vivo studies elaborate numerous different molecular interactions between complement activation products and hemostasis, this review focuses on the clinical evidence that links these 2 fields in humans. Several noninfectious conditions with known complement involvement are scrutinized for common patterns concerning a prothrombotic statues and the occurrence of certain complement activation levels. Next to PNH and aHUS, germline-encoded CD59 or CD55 deficiency (the latter causing the disease complement hyperactivation, angiopathic thrombosis, and protein-losing enteropathy), autoimmune hemolytic anemia, (catastrophic) antiphospholipid syndrome, and C3 glomerulopathy are considered. Parallels and distinct features among these conditions are discussed against the background of thrombosis, complement activation, and potential complement diagnostic and therapeutic avenues.

Complement is one of the most ancient defense systems. It gets strongly activated immediately after acute injuries like trauma, burn, or sepsis and helps to initiate regeneration. However, uncontrolled complement activation contributes to disease progression instead of supporting healing. Such effects are perceptible not only at the site of injury but also systemically, leading to systemic activation of other intravascular cascade systems eventually causing dysfunction of several vital organs. Understanding the complement pathomechanism and its interplay with other systems is a strict requirement for exploring novel therapeutic intervention routes. Ex vivo models exploring the cross-talk with other systems are rather limited, which complicates the determination of the exact pathophysiological roles that complement has in trauma, burn, and sepsis. Literature reporting on these three conditions is often controversial regarding the importance, distribution, and temporal occurrence of complement activation products further hampering the deduction of defined pathophysiological pathways driven by complement. Nevertheless, many in vitro experiments and animal models have shown beneficial effects of complement inhibition at different levels of the cascade. In the future, not only inhibition but also a complement reconstitution therapy should be considered in prospective studies to expedite how meaningful complement-targeted interventions need to be tailored to prevent complement augmented multi-organ failure after trauma, burn, and sepsis.This review summarizes clinically relevant studies investigating the role of complement in the acute diseases trauma, burn, and sepsis with important implications for clinical translation.

Factor H (FH), a member of the regulators-of-complement-activation (RCA) family of proteins, circulates in human plasma at concentrations of 180–420 mg/L where it controls the alternative pathway (AP) of complement in the fluid phase and on cell surfaces. When the regulatory function of FH is impaired, complement-mediated tissue injury and inflammation occur, leading to diseases such as atypical hemolytic uremic syndrome (a thrombotic microangiopathy or TMA), C3 glomerulopathy (C3G) and monoclonal gammopathy of renal significance (MGRS). A pathophysiological cause of compromised FH function is the development of autoantibodies to various domains of the FH protein. FH autoantibodies (FHAAs) are identified in 10.9% of patients with aHUS, 3.2% of patients with C3G, and rarely in patients with MGRS. The phenotypic variability of FHAA-mediated disease reflects both the complexity of FH and the epitope specificity of FHAA for select regions of the native protein. In this paper, we have characterized FHAA epitopes in a large cohort of patients diagnosed with TMA, C3G or MGRS. We explore the epitopes recognized by FHAAs in these diseases and the association of FHAAs with the genetic deletion of both copies of the CFHR1 gene to show how these disease phenotypes are associated with this diverse spectrum of autoantibodies.

To discriminate between self and non-self surfaces and facilitate immune surveillance, the complement system relies on the interplay between surface-directed activators and regulators. The dimeric modulator FHR-1 is hypothesized to competitively remove the complement regulator FH from surfaces that strongly fix opsonic C3b molecules—a process known as “deregulation.” The C-terminal regions of FH and FHR-1 provide the basis of this competition. They contain binding sites for C3b and host surface markers and are identical except for two substitutions: S1191L and V1197A (i.e., FH “SV”; FHR-1 “LA”). Intriguingly, an FHR-1 variant featuring the “SV” combination of FH predisposes to atypical hemolytic uremic syndrome (aHUS). The functional impact of these mutations on complement (de)regulation, and their pathophysiological consequences, have largely remained elusive. We have addressed these questions using recombinantly expressed wildtype, mutated, and truncated versions of FHR-1 and FH. The “SV” to “LA” substitutions did not affect glycosaminoglycan recognition and had only a small effect on C3b binding. In contrast, the two amino acids substantially affected the binding of FH and FHR-1 to α2,3-linked sialic acids as host surfaces markers, with the S-to-L substitution causing an almost complete loss of recognition. Even with sialic acid-binding constructs, notable deregulation was only detected on host and not foreign cells. The aHUS-associated “SV” mutation converts FHR-1 into a sialic acid binder which, supported by its dimeric nature, enables excessive FH deregulation and, thus, complement activation on host surfaces. While we also observed inhibitory activities of FHR-1 on C3 and C5 convertases, the high concentrations required render the physiological impact uncertain. In conclusion, the SV-to-LA substitution in the C-terminal regions of FH and FHR-1 diminishes its sialic acid-binding ability and results in an FHR-1 molecule that only moderately deregulates FH. Such FH deregulation by FHR-1 only occurs on host/host-like surfaces that recruit FH. Conversion of FHR-1 into a sialic acid binder potentiates the deregulatory capacity of FHR-1 and thus explains the pathophysiology of the aHUS-associated FHR-1 “SV” variant.

Triple negative human breast cancer (TNBC) is an aggressive cancer subtype with poor prognosis. Besides the better-known artemisinin, Artemisia annua L. contains numerous active compounds not well-studied yet. High-performance liquid chromatography coupled with diode-array and mass spectrometric detection (HPLC-DAD-MS) was used for the analysis of the most abundant compounds of an Artemisia annua extract exhibiting toxicity to MDA-MB-231 TNBC cells. Artemisinin, 6,7-dimethoxycoumarin, arteannuic acid were not toxic to any of the cancer cell lines tested. The flavonols chrysosplenol d and casticin selectively inhibited the viability of the TNBC cell lines, MDA-MB-231, CAL-51, CAL-148, as well as MCF7, A549, MIA PaCa-2, and PC-3. PC-3 prostate cancer cells exhibiting high basal protein kinase B (AKT) and no ERK1/2 activation were relatively resistant, whereas MDA-MB-231 cells with high basal ERK1/2 and low AKT activity were more sensitive to chrysosplenol d treatment. In vivo, chrysosplenol d and casticin inhibited MDA-MB-231 tumor growth on chick chorioallantoic membranes. Both compounds induced mitochondrial membrane potential loss and apoptosis. Chrysosplenol d activated ERK1/2, but not other kinases tested, increased cytosolic reactive oxygen species (ROS) and induced autophagy in MDA-MB-231 cells. Lysosomal aberrations and toxicity could be antagonized by ERK1/2 inhibition. The Artemisia annua flavonols chrysosplenol d and casticin merit exploration as potential anticancer therapeutics.

Scientists hypothesized that drugs such as ibuprofen or renin-angiotensin system (RAS) blockers could exacerbate the novel coronavirus disease COVID-19 by upregulating the angiotensin-converting enzyme 2 (ACE2), which serves as an entry receptor for the coronavirus SARS-CoV-2. This hypothesis was taken up by the lay press and led to concerns among doctors and patients whether the use of these drugs was still safe and justified against the background of the pandemic spread of SARS-CoV-2 with an increasing number of cases and deaths. In this article, we summarize what is known about the effect of RAS blockers or non-steroidal anti-inflammatory drugs (NSAIDs) on the course of COVID-19 disease. In the case of RAS inhibition, we also find evidence for the opposite hypothesis, namely, that RAS inhibition in COVID-19 could be protective. In view of the inconsistent and limited evidence and after weighing up the benefits and risks, we would not currently recommend discontinuing or switching an effective treatment with RAS blockers. NSAIDs should be used at the lowest effective dose for the shortest possible period. The choice of drug to treat COVID-19-associated fever or pain should be based on a benefit-risk assessment for known side effects (e.g., kidney damage, gastrointestinal ulceration).

Background Therapeutic intervention strategies in complement-mediated hemolytic diseases are still inappropriate, and lethal events cannot be reliably prevented. As an in vitro model of intravascular hemolysis, a sensitive flow cytometric assay was designed using red blood cells (RBCs) of patients with paroxysmal nocturnal hemoglobinuria (PNH) as target cells. Complement activation by human allo- and autoantibodies directed against RBC antigens and the effect of different complement inhibitors were studied. Study design and methods RBCs of patients with a PNH III RBC clone of more than 20% were coated with different human allo- or autoantibodies. Hemolysis was initiated with pooled normal human AB serum with or without the addition of complement inhibitors. Loss of PNH III RBCs was estimated by flow cytometry. Results RBC antibodies of 174 different patients representing 37 different specificities were tested for their potency to activate complement. In correlation with blood group specificities roughly three different patterns were observed: 1) strong and regular, 2) sporadic, and 3) weak or absent complement activation. Remarkably strong complement activators were among antibodies directed against high-prevalence blood group antigens. The C5 inhibitor eculizumab abrogated mild but not strong complement activation, even in presence of excess inhibitor. However, this residual complement activity could be further depressed by combining eculizumab with other inhibitors. Conclusion The PNH hemolysis assay offers a sensitive tool for in vitro analyses of classical pathway-mediated complement activation. The recognition of additive effects of complement inhibitors may guide novel intervention strategies against unwanted complement damage.

During local and systemic inflammation, the complement system and neutrophil granulocytes are activated not only by pathogens, but also by released endogenous danger signals. It is recognized increasingly that complement-mediated neutrophil activation plays an ambivalent role in sepsis pathophysiology. According to the current definition, the onset of organ dysfunction is a hallmark of sepsis. The preceding organ damage can be caused by excessive complement activation and neutrophil actions against the host, resulting in bystander injury of healthy tissue. However, in contrast, persistent and overwhelming inflammation also leads to a reduction in neutrophil responsiveness as well as complement components and thus may render patients at enhanced risk of spreading infection. This review provides an overview on the molecular and cellular processes that link complement with the two-faced functional alterations of neutrophils in sepsis. Finally, we describe novel tools to modulate this interplay beneficially in order to improve outcome.

// Claudia Schmidt 1, 2 , Cornelia Loos 1, 3 , Lu Jin 1 , Michael Schmiech 1 , Christoph Q. Schmidt 1 , Menna El Gaafary 1, 4 , Tatiana Syrovets 1, * and Thomas Simmet 1, * 1 Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, Ulm, Germany 2 Present address: Rommelag CMO, Sulzbach-Laufen, Germany 3 Present address: Institute of Protein Biochemistry, Ulm University, Ulm, Germany 4 Present address: Department of Pharmacognosy, College of Pharmacy, Cairo University, Giza, Egypt * These authors contributed equally to this work Correspondence to: Thomas Simmet, email: thomas.simmet@uni-ulm.de Keywords: triterpenoids, apoptosis, prostate cancer, allosteric Akt inhibitor Received: February 09, 2017 Accepted: June 27, 2017 Published: July 08, 2017 ABSTRACT The triterpenoid acetyl-lupeolic acid (ac-LA) isolated from the oleogum resin of Boswellia carterii reduced the viability of a panel of cancer cell lines more efficiently than lupeol. There was no detectable intracellular conversion of ac-LA to lupeol and vice versa . In contrast to docetaxel, ac-LA did not induce selection of treatment-resistant cancer cells. By various parameters including DNA fragmentation, ac-LA was shown to induce apoptosis in androgen-independent PC-3 cells, whereas in MDA-MB-231 breast cancer cells, ac-LA led to cell accumulation in the G 2 /M phase of the cell cycle, but not to apoptosis. In silico docking combined with in vitro kinase assays implied that ac LA potently inhibits Akt mainly by direct binding to the pleckstrin homology domain. Consistently, an Akt1 mutant deficient of the PH domain afforded partial resistance to ac-LA and complete resistance to lupeol and the Akt inhibitor III. Ac-LA inhibited phosphorylation of downstream targets of the Akt signaling pathway, which was followed by inhibition of the mTOR target p70 ribosomal six protein kinase and the nuclear accumulation of p65/NF-κB, β-catenin, and c-myc, as well as loss of the mitochondrial membrane potential. Ac-LA exhibited antiproliferative, proapoptotic, and antitumorigenic effects on PC-3-tumors xenografted either on chick chorioallantoic membranes or in nude mice. Ac-LA exhibited a clearly better safety profile than docetaxel or lupeol during chronic administration in vivo . In contrast to lupeol, ac-LA also inhibited release of vascular endothelial growth factor in vitro and accordingly angiogenesis in vivo . Thus, ac-LA deserves further exploration as a potential new antitumor compound.

Eculizumab inhibits the terminal, lytic pathway of complement by blocking the activation of the complement protein C5 and shows remarkable clinical benefits in certain complement-mediated diseases. However, several reports suggest that activation of C5 is not always completely suppressed in patients even under excess of eculizumab over C5, indicating that residual C5 activity may derogate the drug's therapeutic benefit under certain conditions. By using eculizumab and the tick-derived C5 inhibitor coversin, we determined conditions ex vivo in which C5 inhibition is incomplete. The degree of such residual lytic activity depended on the strength of the complement activator and the resulting surface density of the complement activation product C3b, which autoamplifies via the alternative pathway (AP) amplification loop. We show that at high C3b densities required for binding and activation of C5, both inhibitors reduce but do not abolish this interaction. The decrease of C5 binding to C3b clusters in the presence of C5 inhibitors correlated with the levels of residual hemolysis. However, by employing different C5 inhibitors simultaneously, residual hemolytic activity could be abolished. The importance of AP-produced C3b clusters for C5 activation in the presence of eculizumab was corroborated by the finding that residual hemolysis after forceful activation of the classical pathway could be reduced by blocking the AP. By providing insights into C5 activation and inhibition, our study delivers the rationale for the clinically observed phenomenon of residual terminal pathway activity under eculizumab treatment with important implications for anti-C5 therapy in general.

Background: Eculizumab blocks the lytic complement pathway by inhibiting C5 and has become the standard of care for certain complement-mediated diseases. Previously, we have shown that strong complement activation in vitro overrides the C5 inhibition by Eculizumab, which accounts for residual terminal pathway activity. Results: Here we show that the levels of residual hemolysis in ex vivo assays differ markedly (up to 3.4-fold) across sera collected from different paroxysmal nocturnal hemoglobinuria (PNH) patients on Eculizumab treatment. This large variability of residual activity was also found in sera of healthy donors, thus cross-validating the findings in patients. While PNH patients with residual lytic activities of 11–30% exhibited hemolysis levels around the upper limit of normal (i.e., plasma LDH of ~250 u/L), as expected for PNH patients on Eculizumab therapy, we found sustained and markedly increased LDH levels of around 400 u/L for the patient with the highest residual activity of 37%. Furthermore, the clinical history of nine out of 14 PNH patients showed intravascular breakthrough hemolysis at the time of documented infections despite ample amounts of administered Eculizumab and/or experimentally determined excess over C5. Conclusion: The occurrence of extraordinary high levels of residual terminal pathway activity in PNH patients receiving Eculizumab is rare, but can impair the suppression of hemolysis. The commonly observed low levels of residual terminal pathway activity seen for most PNH patients can exacerbate during severe infections and, thus, can cause pharmacodynamic breakthrough hemolysis in PNH patients treated with Eculizumab.

Frankincense is an oleogum resin from Boswellia trees of the Burseraceae family. The oleogum resin contains 15–20 % triterpenic acids, including therapeutically active boswellic acids [1]. Extracts...

Background Artemisia annua L. has gained increasing attention for its anticancer activity. However, beside artemisinin, less is known about the possible bioactive ingredients of Artemisia annua and respective herbal preparations. We hypothesized that, in addition to artemisinin, Artemisia annua preparations might contain multiple ingredients with potential anticancer activity. Methods MDA-MB-231 triple negative human breast cancer (TNBC) cells along with other treatment resistant, metastatic cancer cell lines were used to investigate in vitro and in vivo the anticancer efficacy of an Artemisia annua extract marketed as a herbal preparation, which contained no detectable artemisinin (limit of detection = 0.2 ng/mg). The extract was characterized by HPLC-DAD and the most abundant compounds were identified by 1H- and 13C NMR spectroscopy and quantified by UHPLC-MS/MS. Cell viability and various apoptotic parameters were quantified by flow cytometry. In vitro data were validated in two in vivo cancer models, the chick chorioallantoic membrane (CAM) assay and in orthotopic breast cancer xenografts in nude mice. Results The Artemisia annua extract, the activity of which could be enhanced by acetonitrile maceration, inhibited the viability of breast (MDA-MB-231 and MCF-7), pancreas (MIA PaCa-2), prostate (PC-3), non-small cell lung cancer (A459) cells, whereas normal mammary epithelial cells, lymphocytes, and PBMC were relatively resistant to extract treatment. Likewise, the extract's most abundant ingredients, chrysosplenol D, arteannuin B, and casticin, but not arteannuic acid or 6,7-dimethoxycoumarin, inhibited the viability of MDA-MB-231 breast cancer cells. The extract induced accumulation of multinucleated cancer cells within 24 h of treatment, increased the number of cells in the S and G2/M phases of the cell cycle, followed by loss of mitochondrial membrane potential, caspase 3 activation, and formation of an apoptotic hypodiploid cell population. Further, the extract inhibited cancer cell proliferation, decreased tumor growth, and induced apoptosis in vivo in TNBC MDA-MB-231 xenografts grown on CAM as well as in nude mice. Conclusion An extract of an artemisinin-deficient Artemisia annua herbal preparation exhibits potent anticancer activity against triple negative human breast cancer. New active ingredients of Artemisia annua extract with potential anticancer activity have been identified.

Mutations in the gene encoding for complement regulator factor H (FH) severely disrupt its normal function to protect human cells from unwanted complement activation, resulting in diseases such as atypical hemolytic uremic syndrome (aHUS). aHUS presents with severe hemolytic anemia, thrombocytopenia, and renal disease, leading to end-stage renal failure. Treatment of severe complement-mediated disease, such as aHUS, by inhibiting the terminal complement pathway, has proven to be successful but at the same time fails to preserve the protective role of complement against pathogens. To improve complement regulation on human cells without interfering with antimicrobial activity, we identified an anti-FH monoclonal antibody (mAb) that induced increased FH-mediated protection of primary human endothelial cells from complement, while preserving the complement-mediated killing of bacteria. Moreover, this FH-activating mAb restored complement regulation in sera from aHUS patients carrying various heterozygous mutations in FH known to impair FH function and dysregulate complement activation. Our data suggest that FH normally circulates in a less active conformation and can become more active, allowing enhanced complement regulation on human cells. Antibody-mediated potentiation of FH may serve as a highly effective approach to inhibit unwanted complement activation on human cells in a wide range of hematological diseases while preserving the protective role of complement against pathogens. ispartof: BLOOD ADVANCES vol:3 issue:4 pages:621-632 ispartof: location:United States status: published

The complement cascade is an ancient immune-surveillance system that not only provides protection from pathogen invasion but has also evolved to participate in physiological processes to maintain tissue homeostasis. The alternative pathway (AP) of complement activation is the evolutionarily oldest part of this innate immune cascade. It is unique in that it is continuously activated at a low level and arbitrarily probes foreign, modified-self, and also unaltered self-structures. This indiscriminate activation necessitates the presence of preformed regulators on autologous surfaces to spare self-cells from the undirected nature of AP activation. Although the other two canonical complement activation routes, the classical and lectin pathways, initiate the cascade more specifically through pattern recognition, their activity still needs to be tightly controlled to avoid excessive reactivity. It is the perpetual duty of complement regulators to protect the self from damage inflicted by inadequate complement activation. Here, we review the role of complement regulators as preformed mediators of defense, explain their common and specialized functions, and discuss selected cases in which alterations in complement regulators lead to disease. Finally, rational engineering approaches using natural complement inhibitors as potential therapeutics are highlighted.

The Ornithodoros moubata Complement Inhibitor (OmCI) binds complement component 5 (C5) with high affinity and, thus, selectively prevents proteolytic activation of the terminal lytic complement pathway. A recombinant version of OmCI (also known as Coversin and rEV576) has proven efficacious in several animal models of complement-mediated diseases and successfully completed a phase Ia clinical trial. Coversin is a small 17 kDa lipocalin protein which has a very short plasma half-life if not bound to C5; therefore, the drug requires frequent dosing. We have improved the pharmacokinetics of Coversin by N-terminal translational conjugation with a 600 residue polypeptide composed of Pro, Ala, and Ser (PAS) residues. To this end, PAS-Coversin as well as the unmodified Coversin were functionally expressed in the cytoplasm of E. coli and purified to homogeneity. Both versions showed identical affinity to human C5, as determined by surface plasmon resonance measurements, and revealed similar complement inhibitory activity, as measured in ELISAs with human serum. In line with the PEG-like biophysical properties, PASylation dramatically prolonged the plasma half-life of uncomplexed Coversin by a factor ≥50 in mice. In a clinically relevant in vitro model of the complement-mediated disease paroxysmal nocturnal hemoglobinuria (PNH) both versions of Coversin effectively reduced erythrocyte lysis. Unexpectedly, while the IC50 values were comparable, PAS-Coversin reached a substantially lower plateau of residual lysis at saturating inhibitor concentrations. Taken together, our data demonstrate two clinically relevant improvements of PASylated Coversin: markedly increased plasma half-life and considerably reduced background hemolysis of erythrocytes with PNH-induced phenotype.

The human complement system is the frontline defense mechanism against invading pathogens. The coexistence of humans and microbes throughout evolution has produced ingenious molecular mechanisms by which microorganisms escape complement attack. A common evasion strategy used by diverse pathogens is the hijacking of soluble human complement regulators to their surfaces to afford protection from complement activation. One such host regulator is factor H (FH), which acts as a negative regulator of complement to protect host tissues from aberrant complement activation. In this report, we show that Plasmodium falciparum merozoites, the invasive form of the malaria parasites, actively recruit FH and its alternative spliced form FH-like protein 1 when exposed to human serum. We have mapped the binding site in FH that recognizes merozoites and identified Pf92, a member of the six-cysteine family of Plasmodium surface proteins, as its direct interaction partner. When bound to merozoites, FH retains cofactor activity, a key function that allows it to downregulate the alternative pathway of complement. In P. falciparum parasites that lack Pf92, we observed changes in the pattern of C3b cleavage that are consistent with decreased regulation of complement activation. These results also show that recruitment of FH affords P. falciparum merozoites protection from complement-mediated lysis. Our study provides new insights on mechanisms of immune evasion of malaria parasites and highlights the important function of surface coat proteins in the interplay between complement regulation and successful infection of the host.

The serum proteins factor H (FH), consisting of 20 complement control protein modules (CCPs), and its splice product FH-like protein 1 (FHL-1; consisting of CCPs 1–7) are major regulators of the alternative pathway (AP) of complement activation. The engineered version of FH, miniFH, contains only the N- and C-terminal portions of FH linked by an optimized peptide and shows ∼10-fold higher ex vivo potency. We explored the hypothesis that regulatory potency is enhanced by unmasking of a ligand-binding site in the C-terminal CCPs 19–20 that is cryptic in full-length native FH. Therefore, we produced an FH variant lacking the central domains 10–15 (FHΔ10–15). To explore how avidity affects regulatory strength, we generated a duplicated version of miniFH, termed midiFH. We compared activities of FHΔ10–15 and midiFH to miniFH, FH, and FHL-1. Relative to FH, FHΔ10–15 exhibited an altered binding profile toward C3 activation products and a 5-fold-enhanced complement regulation on a paroxysmal nocturnal hemoglobinuria patient’s erythrocytes. Contrary to dogma, FHL-1 and FH exhibited equal regulatory activity, suggesting that the role of FHL-1 in AP regulation has been underestimated. Unexpectedly, a substantially increased avidity for complement opsonins, as seen in midiFH, did not potentiate the inhibitory potential on host cells. In conclusion, comparisons of engineered and native FH-based regulators have identified features that determine high AP regulatory activity on host cells. Unrestricted availability of FH CCPs 19–20 and an optimal spatial orientation between the N- and C-terminal FH regions are key.

Complement factor H (FH), an elongated and substantially glycosylated 20-domain protein, is a soluble regulator of the complement alternative pathway (AP). It contains several glycan binding sites which mediate recognition of α2-3-linked sialic acid (FH domain 20) and glycosaminoglycans (domains 6-8 and 19-20). FH also binds the complement C3-activation product C3b, a powerful opsonin and focal point for the formation of C3-convertases of the AP feedback loop. In freely circulating FH the C3b binding site in domains 19-20 is occluded, a phenomenon that is not fully understood and could be mediated by an intramolecular interaction between FH's intrinsic sialylated glycosylation and its own sialic acid binding site. In order to assess this possibility, we characterized FH's sialylation with respect to glycosidic linkage type and searched for further potential, not yet characterized sialic acid binding sites in FH and its seven-domain spanning splice variant and fellow complement regulator FH like-1 (FHL-1). We also probed FH binding to the sialic acid variant Neu5Gc which is not expressed in humans but on heterologous erythrocytes that restrict the human AP and in FH transgenic mice. We find that FH contains mostly α2-6-linked sialic acid, making an intramolecular interaction with its α2-3-sialic acid specific binding site and an associated self-lock mechanism unlikely, substantiate that there is only a single sialic acid binding site in FH and none in FHL-1, and demonstrate direct binding of FH to the nonhuman sialic acid Neu5Gc, supporting the use of FH transgenic mouse models for studies of complement-related diseases.

Malaria parasites have evolved ingenious mechanisms to escape the human immune system to enable the establishment of successful blood-stage infection. Recent studies identifying novel host-pathogen interactions between the complement system and malaria parasites yield insights on the mechanisms of parasite entry into red blood cells, complement evasion strategies and the development of malaria pathogenesis. In particular, human complement regulators are co-opted by parasite adhesins and surface proteins for parasite invasion and to evade complement attack. However, these studies are also beginning to reveal the duality of the complement system in mediating both parasite destruction and facilitating efficient parasite invasion. The following provides a brief overview of complement evasion strategies employed by blood-stage malaria parasites and highlights the importance of understanding the complex interplay between complement and parasite infection.

Vivax malaria host receptor Human malaria is caused by half a dozen species of Plasmodium protozoan parasites, each with distinctive biology. P. vivax , which causes relapsing malaria, specifically parasitizes immature red blood cells called reticulocytes. Gruszczyk et al. identified TfR1 (host transferrin receptor 1) as an alternative receptor for P. vivax . TfR1 binds to a specific P. vivax surface protein. However, the parasite that causes cerebral malaria, P. falciparum , does not share TfR1 as a receptor: P. falciparum could still infect cells in which TfR1 expression was knocked down, but P. vivax could not. Monoclonal antibodies to the P. vivax protein successfully hindered P. vivax infection of red blood cells. Science , this issue p. 48

The complement system labels microbes and host debris for clearance. Degradation of surface-bound C3b is pivotal to direct immune responses and protect host cells. How the serine protease factor I (FI), assisted by regulators, cleaves either two or three distant peptide bonds in the CUB domain of C3b remains unclear. We present a crystal structure of C3b in complex with FI and regulator factor H (FH; domains 1-4 with 19-20). FI binds C3b-FH between FH domains 2 and 3 and a reoriented C3b C-terminal domain and docks onto the first scissile bond, while stabilizing its catalytic domain for proteolytic activity. One cleavage in C3b does not affect its overall structure, whereas two cleavages unfold CUB and dislodge the thioester-containing domain (TED), affecting binding of regulators and thereby determining the number of cleavages. These data explain how FI generates late-stage opsonins iC3b or C3dg in a context-dependent manner, to react to foreign, danger or healthy self signals.

Plasmodium vivax shows a strict host tropism for reticulocytes. We identify transferrin receptor 1 (TfR1) as the receptor for P. vivax reticulocyte-binding protein 2b (PvRBP2b). The structure of the N-terminal domain of PvRBP2b involved in red blood cell binding was determined, elucidating the molecular basis for TfR1 recognition. TfR1 was validated as the biological target of PvRBP2b engagement by TfR1 expression knockdown analysis. TfR1 mutant cells deficient in PvRBP2b binding were refractory to invasion of P. vivax, but not to invasion of P. falciparum. Using Brazilian and Thai clinical isolates, we show that PvRBP2b monoclonal antibodies that inhibit reticulocyte binding also block P. vivax entry into reticulocytes. These data show that TfR1-PvRBP2b invasion pathway is critical for the recognition of reticulocytes during P. vivax invasion.

The tight regulation of innate immunity on extracellular matrix (ECM) is a vital part of immune homeostasis throughout the human body, and disruption to this regulation in the eye is thought to contribute directly to the progression of age-related macular degeneration (AMD). The plasma complement regulator factor H (FH) is thought to be the main regulator that protects ECM against damaging complement activation. However, in the present study we demonstrate that a truncated form of FH, called FH-like protein 1 (FHL-1), is the main regulatory protein in the layer of ECM under human retina, called Bruch’s membrane. Bruch’s membrane is a major site of AMD disease pathogenesis and where drusen, the hallmark lesions of AMD, form. We show that FHL-1 can passively diffuse through Bruch’s membrane, whereas the full sized, glycosylated, FH cannot. FHL-1 is largely bound to Bruch’s membrane through interactions with heparan sulfate, and we show that the common Y402H polymorphism in the CFH gene, associated with an increased risk of AMD, reduces the binding of FHL-1 to this heparan sulfate. We also show that FHL-1 is retained in drusen whereas FH coats the periphery of the lesions, perhaps inhibiting their clearance. Our results identify a novel mechanism of complement regulation in the human eye, which highlights potential new avenues for therapeutic strategies.

In this issue of Blood, Keshari et al report on the striking absence of substantial complement system activation in a model of generalized experimental coagulation and fibrinolysis in baboons.1

A novel series of imidazol-5-yl carbinols and their 4-chlorobenzoyl esters has been synthesized by the Grignard reaction and subsequent esterification. These compounds were screened for their antimicrobial activities in an agar diffusion assay. The compounds with C10 to C12-alkyl side chains displayed significant antimycotic activity.

After severe trauma, the immune system is challenged with a multitude of endogenous and exogenous danger molecules. The recognition of released danger patterns is one of the prime tasks of the innate immune system. In the last two decades, numerous studies have established the complement cascade as a major effector system that detects and processes such danger signals. Animal models with engineered deficiencies in certain complement proteins have demonstrated that widespread complement activation after severe injury culminates in complement dysregulation and excessive generation of complement activation fragments. Such exuberant pro-inflammatory signaling evokes systemic inflammation, causes increased susceptibility to infections and is associated with a detrimental course of the disease after injury. We discuss the underlying processes of such complementopathy and recapitulate different intervention strategies within the complement cascade. So far, several orthogonal anti-complement approaches have been tested with varying success in a large number of rodent, in several porcine and few simian studies. We illustrate the different features among those intervention strategies and highlight those that hold the greatest promise to become potential therapeutic options for the intricate disease of traumatic injury.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated cell lysis due to deficiency of GPI-anchored complement regulators. Blockage of the lytic pathway by eculizumab is the only available therapy for PNH patients and shows remarkable benefits, but regularly yields PNH erythrocytes opsonized with fragments of complement protein C3, rendering such erythrocytes prone to extravascular hemolysis. This effect is associated with insufficient responsiveness seen in a subgroup of PNH patients. Novel C3-opsonin targeted complement inhibitors act earlier in the cascade, at the level of activated C3 and are engineered from parts of the natural complement regulator Factor H (FH) or complement receptor 2 (CR2). This inhibitor class comprises three variants of "miniFH" and the clinically developed "FH-CR2" fusion-protein (TT30). We show that the approach of FH-CR2 to target C3-opsonins was more efficient in preventing complement activation induced by foreign surfaces, whereas the miniFH variants were substantially more active in controlling complement on PNH erythrocytes. Subtle differences were noted in the ability of each version of miniFH to protect human PNH cells. Importantly, miniFH and FH-CR2 interfered only minimally with complement-mediated serum killing of bacteria when compared to untargeted inhibition of all complement pathways by eculizumab. Thus, the molecular design of each C3-opsonin targeted complement inhibitor determines its potency in respect to the nature of the activator/surface providing potential functionality in PNH.

Factor H autoantibodies are found in ~10% of aHUS patients. Most are associated with complete deficiency of factor H related proteins 1/3 and bind to the C terminal recognition domain. MPGN, like aHUS, is characterised by complement activation. In this study we, therefore, examined the hypothesis that factor H autoantibodies are associated with MPGN. We screened sera from 16 MPGN patients and 100 normal controls using ELISA and detected strongly positive IgG factor H autoantibodies in 2 patients. One patient had type II (DDD) MPGN (male aged 24 yrs) with C3NeF and the other type I (female aged 26 yrs) with no detectable C3NeF. We identified the binding site of the autoantibodies using small SCR domain fragments in the ELISA and showed that the autoantibodies in both patients bound predominately to the N terminal complement regulatory domain of factor H. We measured CFHR 1/3 copy number using MLPA and showed that both patients had 2 copies of CFHR1 and 3. Finally, we examined the functionality of detected factor H autoantibodies using purified patient IgG and observed increased haemolysis when purified IgG from both patients was added to normal human sera prior to incubation with rabbit red blood cells. Thus, in a cohort of MPGN patients we have found a high titre of functionally significant factor H autoantibodies in two patients with MPGN. Antibody depleting therapy may have a role in such patients and we suggest that screening for factor H autoantibodies should be undertaken in all patients with MPGN.

The Plasmodium falciparum adhesin PfRh4 binds to complement receptor type-1 (CR1) on human erythrocytes and mediates a glycophorin-independent invasion pathway. CR1 is a complement regulator and immune-adherence receptor on erythrocytes required for shuttling of C3b/C4b-opsonized particles to liver and spleen for phagocytosis. Using recombinant CR1 constructs, we mapped the recognition site for PfRh4 to complement control protein modules 1 to 3 (CCP1-3) at the membrane-distal amino terminus of CR1. This region of CR1 binds to C4b and C3b and accelerates decay of both classic pathway and alternative pathway C3 and C5 convertases. CCP1-3 competed for PfRh4 binding to erythroid CR1 and inhibited the PfRh4-CR1 invasion pathways across a wide range of P falciparum strains. PfRh4 did not bind significantly to other CR1 constructs, including CCP15-17, which is 85% identical to CCP1-3. PfRh4 binding to CR1 did not affect its C3b/C4b binding capability, and we show evidence for a ternary complex between CCP1-3, C4b, and PfRh4. PfRh4 binding specifically inhibited CR1's convertase decay-accelerating activity, whereas there was no effect on factor H-mediated decay-accelerating activity. These results increase our understanding of the functional implications of CR1 engagement with PfRh4 and highlight the interplay between complement regulation and infection.

Human complement factor H (FH), an abundant 155-kDa plasma glycoprotein with 40 disulphide bonds, regulates the alternative-pathway complement cascade. Mutations and single nucleotide polymorphisms in the FH gene predispose to development of age-related macular degeneration, atypical haemolytic uraemic syndrome and dense deposit disease. Supplementation with FH variants protective against disease is an enticing therapeutic prospect. Current sources of therapeutic FH are restricted to human blood plasma highlighting a need for recombinant material. Previously FH expression in cultured plant, mammalian or insect cells yielded protein amounts inadequate for full characterisation, and orders of magnitude below therapeutic usefulness. Here, the V62,Y402 variant of FH has been produced recombinantly (rFH) in Pichia pastoris cells. Codon-optimisation proved essential whilst exploitation of the yeast mating α-factor peptide ensured secretion. We thereby produced multiple 10s-of-milligram of rFH. Following endoglycosidase H digestion of N-linked glycans, rFH (with eight residual N-acetylglucosamine moieties) was purified on heparin-affinity resin and anion-exchange chromatography. Full-length rFH was verified by mass spectrometry and Western blot using monoclonal antibodies to the C-terminus. Recombinant FH is a single non-aggregated species (by dynamic light scattering) and fully functional in biochemical and biological assays. An additional version of rFH was produced in which eight N-glycosylation sequons were ablated by Asn-Gln substitutions resulting in a glycan-devoid product. Successful production of rFH in this potentially very highly expressing system makes production of therapeutically useful quantities economically viable. Furthermore, ease of genetic manipulation in P. pastoris would allow production of engineered FH versions with enhanced pharmacokinetic and pharmacodynamic properties.