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The complement system plays a key role in opsonization and immune clearance of engineered nanoparticles. Understanding the efficiency, inter-subject, and inter-strain differences of complement opsonization in preclinical species can help with translational nanomedicine development and improve our ability to model complement response in humans. Dextran-coated superparamagnetic iron oxide (SPIO) nanoparticles and a wide range of non-magnetic iron oxide nanoparticle formulations are widely used in magnetic resonance imaging and as clinically approved iron supplements. Previously we found that opsonization of SPIO nanoworms (NW) with the third complement protein (C3) proceeds mostly via the alternative pathway in humans, and via the lectin pathway in mice. Here, we studied the pathway and efficiency of opsonization of 106 nm SPIO NW with C3 in different preclinical species and commonly used laboratory strains. In sera of healthy human donors (n = 6), C3 opsonization proceeded exclusively through the alternative pathway. On the other hand, the C3 opsonization in dogs (6 breeds), rats (4 strains) and mice (5 strains) sera was either partially or completely dependent on the complement Ca2+-sensitive pathways (lectin and/or classical). Specifically, C3 opsonization in sera of Long Evans rat strain, and mouse strains widely used in nanomedicine research (BALB/c, C57BL/6 J, and A/J) was only through the Ca2+-dependent pathways. Dogs and humans had the highest between-subject variability in C3 opsonization levels, while rat and mouse sera showed the lowest between-strain variability. Furthermore, using a panel of SPIO nanoparticles of different sizes and dextran coatings, we found that the level of C3 opsonization (C3 molecules per milligram Fe) in human sera was lower than in animal sera. At the same time, there was a strong predictive value of complement opsonization in dog and rat sera; nanoparticles with higher C3 deposition in animals showed higher deposition in humans, and vice versa. Notably, the opsonization decreased with decreasing size in all sera. The studies highlight the importance of the consideration of species and strains for predicting human complement responses (opsonization) towards nanomedicines.

Semen is important in determining HIV-1 susceptibility but it is unclear how it affects virus transmission during sexual contact. Mucosal Langerhans cells (LCs) are the first immune cells to encounter HIV-1 during sexual contact and have a barrier function as LCs are restrictive to HIV-1. As semen from people living with HIV-1 contains complement-opsonized HIV-1, we investigated the effect of complement on HIV-1 dissemination by human LCs in vitro and ex vivo. Notably, pre-treatment of HIV-1 with semen enhanced LC infection compared to untreated HIV-1 in the ex vivo explant model. Infection of LCs and transmission to target cells by opsonized HIV-1 was efficiently inhibited by blocking complement receptors CR3 and CR4. Complement opsonization of HIV-1 enhanced uptake, fusion, and integration by LCs leading to an increased transmission of HIV-1 to target cells. However, in the absence of both CR3 and CR4, C-type lectin receptor langerin was able to restrict infection of complement-opsonized HIV-1. These data suggest that complement enhances HIV-1 infection of LCs by binding CR3 and CR4, thereby bypassing langerin and changing the restrictive nature of LCs into virus-disseminating cells. Targeting complement factors might be effective in preventing HIV-1 transmission.

Background & Aims Liver CRIg+ (complement receptor of the immunoglobulin superfamily) macrophages play a critical role in filtering bacteria and their products from circulation. Translocation of microbiota-derived products from an impaired gut barrier contributes to the development of obesity-associated tissue inflammation and insulin resistance. However, the critical role of CRIg+ macrophages in clearing microbiota-derived products from the bloodstream in the context of obesity is largely unknown. Methods We performed studies with CRIg–/–, C3–/–, cGAS–/–, and their wild-type littermate mice. The CRIg+ macrophage population and bacterial DNA abundance were examined in both mouse and human liver by either flow cytometric or immunohistochemistry analysis. Gut microbial DNA–containing extracellular vesicles (mEVs) were adoptively transferred into CRIg–/–, C3–/–, or wild-type mice, and tissue inflammation and insulin sensitivity were measured in these mice. After coculture with gut mEVs, cellular insulin responses and cGAS/STING-mediated inflammatory responses were evaluated. Results Gut mEVs can reach metabolic tissues in obesity. Liver CRIg+ macrophages efficiently clear mEVs from the bloodstream through a C3-dependent opsonization mechanism, whereas obesity elicits a marked reduction in the CRIg+ macrophage population. Depletion of CRIg+ cells results in the spread of mEVs into distant metabolic tissues, subsequently exacerbating tissue inflammation and metabolic disorders. Additionally, in vitro treatment of obese mEVs directly triggers inflammation and insulin resistance of insulin target cells. Depletion of microbial DNA blunts the pathogenic effects of intestinal EVs. Furthermore, the cGAS/STING pathway is crucial for microbial DNA–mediated inflammatory responses. Conclusions Deficiency of CRIg+ macrophages and leakage of intestinal EVs containing microbial DNA contribute to the development of obesity-associated tissue inflammation and metabolic diseases.

In efforts to achieve minimal systemic toxicity and high tumor delivery efficiencies in cancer therapy, various nanomedicine formulations having stealth polymer coatings have been developed for minimizing immune cell uptake and off-target macrophage phagocyte system (MPS) organ accumulation. Despite an initial reduction in immune cell uptake, stealth nanoparticles still initiate an antibody immune response. This response acts on subsequent administrations in treatment regimens resulting in accelerated blood clearance of particles into MPS organs, particularly the liver, where they are retained for prolonged periods. Consequently, doses after the first administration in treatment regimens have diminished tumor accumulation and increased MPS toxicity. Here, we present a strategy reducing antibody responses to each dose in a treatment regimen by alternating between polyethylene-glycol and polymethyloxazoline polymers as the nanoparticle coating between administrations. In a weekly dosing regimen, we find that the first dose of particles having either coating display similar favorable pharmacokinetics and biodistributions, thus allowing the polymers to be used interchangeably. However, when maintaining the same coating in subsequent administrations, we find that particles are in circulation at the height of the antibody immune response resulting in 50-60% decreases of circulation half-lives and tumor accumulation along with 50% increases in liver accumulation. By alternating the polymers used in the nanoparticle coating between administrations, we find each dose maintains favorable in vivo behaviors at the height of the antibody immune response to the previous administration. Furthermore, our strategy increases the clearance of particles uptaken by macrophages and hepatocytes, resulting in marked decreases in hepatotoxicity.

Vaccination evoking immunity in susceptible individuals has become the most effective solution to combat infectious diseases. The surface antigen of hepatitis B virus (HBsAg) is a mandatory vaccine for children in China. Herein, we designed an antimicrobial protein consisting of an antimicrobial peptide Thanatin at the N-terminus fused with the HBsAg at the C-terminus. The expressed Thanatin-GFP-HBsAg (TGH) quantitively bound with the anti-HBsAg antibody by ELISA, and after exposure to TGH, Gram-negative E. coli cells became fluorescencent indicating the binding of TGH with the bacterial cells. We also demonstrated that TGH could intercalated into the lipid bilayer as shown by the quartz crystal microbalance with dissipation (QCM-D) and TEM. Moreover, the TGH bound E. coli cells attracted anti-HBsAg IgG as shown by the experiments that in turn treated the E. coli cells with TGF, anti-HBsAg serum and PE labelled goat anti-mouse IgG antibodies. After supplementation with serum from HBsAg vaccinated individuals, TGH showed improved bactericidal effect in vitro. In vivo experiments showed that the mice receiving TGH vaccination show quicker clearance of MDR E. coli pretreated with TGH and better survive in comparison with groups treated with piperacillin plus subatan. In addition, anti-HBsAg serum supplementation also improved the endocytosis of TGH decorated bacteria by leukocytes. This study reported a novel solution to combat infectious pathogens based on the membrane penetrating effect of antimicrobial peptides.

Cationic liposomes (CLs) have been regarded as the most promising gene delivery vectors for decades with the advantages of excellent biodegradability, biocompatibility, and high nucleic acid encapsulation efficiency. However, the clinical use of CLs in cancer gene therapy is limited because of many uncertain factors in vivo. Extracellular barriers such as opsonization, rapid clearance by the reticuloendothelial system and poor tumor penetration, and intracellular barriers, including endosomal/lysosomal entrapped network and restricted diffusion to the nucleus, make CLs not the ideal vector for transferring extrinsic genes in the body. However, the obstacles in achieving productive therapeutic effects of nucleic acids can be addressed by tailoring the properties of CLs, which are influenced by lipid compositions and surface modification. This review focuses on the physiological barriers of CLs against cancer gene therapy and the effects of lipid compositions on governing transfection efficiency, and it briefly discusses the impacts of particle size, membrane charge density, and surface modification on the fate of CLs in vivo, which may provide guidance for their preclinical studies., Graphical Abstract, The concept of lipoplexes applied to cancer gene therapy has been popular for decades; however, no such medicine has been marketed. Deng and colleagues highlight the physiological barriers against the clinical success of the cationic liposomal gene delivery system in cancer therapy. They suggest that the rational design of clinically applicable lipoplexes requires designers to pay close attention to the barriers and the impact of physicochemical properties of cationic liposomes on the therapeutic outcome.

The complement system consists of a collection of serum proteins that act as the main frontline effector arm of the innate immune system. Activation of complement can occur through 3 individual induction pathways: the classical, mannose-binding lectin, and alternative pathways. Activation results in opsonization, recruitment of effector cells through potent immune mediators known as anaphylatoxins, and cell lysis via the formation of the membrane attack complex. Stringent regulation of complement is required to protect against inappropriate activation of the complement cascade. Complement activation within the tumor microenvironment does not increase antitumoral action; instead, it enhances tumor growth and disease progression. Radiation therapy (RT) is a staple in the treatment of malignancies and controls tumor growth through direct DNA damage and the influx of immune cells, reshaping the makeup of the tumor microenvironment. The relationship between RT and complement activity in the tumor microenvironment is uncertain at best. The following review will focus on the complex interaction of complement activation and the immune-modulating effects of RT and the overall effect on tumor progression. The clinical implications of complement activation in cancer and the use of therapeutics and potential biomarkers will also be covered.

Background Platelet refractoriness remains a challenging clinical dilemma although significant advancements have been made in identifying human leukocyte antigen (HLA) matched or HLA compatible units. Antiplatelet antibodies are the major risk factor for immune-mediated platelet refractoriness, yet the role of antibody-initiated complement-mediated platelet destruction remains poorly understood. Study Design and Methods Human complement-mediated opsonization and killing of platelets was assayed ex vivo using antibody-sensitized human platelets incubated with complement-sufficient human sera. A new animal model of platelet refractoriness utilizing Wistar rats transfused with human platelets is described. Results Human platelets sensitized with anti-platelet antibodies were rapidly opsonized with iC3b upon incubation in human sera. This opsonization could be completely blocked with a classical pathway complement inhibitor, PA-dPEG24. Complement activation decreased platelet viability, which was also reversible with complement inhibitor PA-dPEG24. A new rat model of platelet refractoriness was developed that demonstrated some platelet removal from the blood stream was complement mediated. Conclusions Complement activation initiated by anti-platelet antibodies leads to complement opsonization and decreased platelet viability. A new rat model of platelet refractoriness was developed that adds a new tool for elucidating the mechanisms of platelet refractoriness.

Polysaccharide antigens do not promote antibody class switching and memory antibody response, thus require conjugation with a T cell dependent carrier protein to generate protective immune response. The intensity of immune responses varies with the carrier proteins for the same carbohydrate antigen and most of the carrier proteins do not generate strong immune responses. Vi polysaccharide and r-flagellin of Salmonella typhi were conjugated and formulated in PLA particles as nanoglycoconjugate which not only generated strong immune response but also promoted antibody class switching and elicited memory antibody response from single point immunization. Nanoglycoconjugate immunization also modulate anti-inflammatory property of Vi polysaccharide with an enhance secretion of pro-inflammatory cytokine TNF-α and IL-6. This was with concomitant decrease of IFN-γ production, antibody class switching from IgG3 to IgG2 with memory antibody generation against Vi polysaccharide. Antibody elicited by nanoglycoconjugate showed better opsonization and clearance of Salmonella typhi in THP-1 macrophages as compared to Vi-flagellin glycoconjugate and Vi TT (Typhbar®). Delivery of glycoconjugate through nanoparticles provides a platform technology for improving the immunogenicity of polysaccharide based vaccines.

Enterococcus faecalis forms single and mixed-species biofilms on both tissue and medical devices in the host, often under exposure to fluid flow, giving rise to infections that are recalcitrant to treatment. The factors that drive enterococcal biofilm formation in the host, however, remain unclear. Recent reports in other pathogens show how surface sensing by bacteria can trigger the transition from planktonic to sessile lifestyle. Fluid flow can enhance initial adhesion, but also influence quorum sensing. Biofilm-specific factors, as well as biofilm size and extracellular polymeric substances, can compromise opsonization and phagocytosis. Bacterial interspecies synergy can create favorable conditions in the host for biofilm formation. Through these concepts, we define the knowledge gaps in understanding host-associated E. faecalis biofilm formation and propose a roadmap for future investigations.

Considering the multifaceted protective and homeostatic roles of the complement system, many consequences arise when drug carriers, and particulate pharmaceutical formulations clash with complement proteins, and trigger complement cascade. Complement activation may induce formulation destabilization, promote opsonization, and affect biological and therapeutic performance of pharmaceutical nano- and micro-particles. In some cases, complement activation is beneficial, where complement may play a role in prophylactic protection, whereas uncontrolled complement activation is deleterious, and contributes to disease progression. Accordingly, design initiatives with particulate medicines should consider complement activation properties of the end formulation within the context of administration route, dosing, systems biology, and therapeutic perspective. Here we examine current progress in mechanistic processes underlying complement activation by pre-clinical and clinical particles, identify opportunities and challenges ahead, and suggest future directions in nanomedicine-complement interface research.

Phagocytosis constitutes a conserved cellular process for multicellular animals to ingest or engulf other cells or particles, which is facilitated by the use of opsonins to bind foreign particles and interact with cell surface receptors. The invertebrate secreted C1q domain-containing proteins (C1qDCs) have been reported to exhibit opsonic activity, while the detailed mechanisms of opsonization still remain unclear. In the present study, a C1qDC (designated as CgC1qDC-5) with opsonic activity was identified from the hemolymph of oyster Crassostrea gigas. CgC1qDC-5 exhibited the ability to bind pathogen-associated molecular patterns (PAMPs) of lipopolysaccharides (LPS) and Lipid A. It could also bind and agglutinate Gram-negative bacteria Escherichia coli, Vibrio splendidus and Vibrio anguillarum, whereas the agglutinating activity could be inhibited by LPS. In addition, CgC1qDC-5 could enhance the phagocytosis of hemocytes toward E. coli, V. splendidus, and V. anguillarum. GST pull-down and surface plasmon resonance assays in vitro revealed that CgC1qDC-5 could interact with β-integrin (CgIntegrin). In vivo, CgC1qDC-5 was observed to bind hemocytes and co-localized with CgIntegrin on the cell membrane of hemocytes. Antibody-mediated blockage of CgIntegrin hindered the CgC1qDC-5-enhanced hemocytic phagocytosis. CgIntegrin also exhibited the ability to bind the Gram-negative bacteria E. coli, V. splendidus, V. anguillarum and Vibrio parahaemolyticus, and PAMP of LPS, but not Lipid A. A phagocytosis assay demonstrated that CgIntegrin could directly mediate phagocytosis toward bacteria as a phagocytic receptor. These results collectively suggested that CgC1qDC-5 could serve as an opsonin to recognize and bind bacteria, and subsequently interact with CgIntegrin on the hemocyte surface to enhance the CgIntegrin-mediated phagocytosis in oyster.

Neutrophils, the most abundant white blood cell, play a critical role in anti-pathogen immunity via phagocytic clearance, secretion of enzymes and immunomodulators, and the release of extracellular traps. Neutrophils non-specifically sense infection through an array of innate immune receptors and inflammatory sensors, but are also able to respond in a pathogen/antigen-specific manner when leveraged by antibodies via Fc-receptors. Among neutrophil functions, antibody-dependent neutrophil phagocytosis (ADNP) results in antibody-mediated opsonization, enabling neutrophils to sense and respond to infection in a pathogen-appropriate manner. Here, we describe a high-throughput flow cytometric approach to effectively visualize and quantify ADNP and its downstream consequences. The assay is easily adaptable, supporting both the use of purified neutrophils or white blood cells, the use of purified Ig or serum, and the broad utility of any target antigen. Thus, this ADNP assay represents a high-throughput platform for the in-depth characterization of neutrophil function., Graphical abstract Unlabelled Image, Highlights • A high-throughput antibody-dependent neutrophil phagocytosis (ADNP) assay was developed. • This flow cytometry assay is flexible and can be easily adapted to any pathogen. • Analysis of sample sets by ADNP assay is fast, robust and cost-effective. • Additional neutrophil functions can be profiled in secondary analyses.

Liposome as a revolutionizing carrier, has exhibited favorable aspects in drug delivery. However, there are numerous challenges affecting its effectiveness. Hypersensitivity, accelerated blood clearance, opsonization, uptake by reticulo-endothelial system, and also immune suppression are known as the dominant manifestations of immune-related effects. They may cause failure to achieve the proof of regulatory approvals. In this review, liposome challenges in biologic environment have been discussed. Moreover, opsonization and liposome interactions with innate and acquired immune system have been investigated. Immune system elements including the corresponding receptors and cells were reviewed in this regard. By exploring immune related interactions of the liposome, it could be possible for the formulators to be aware of the immune related interactions of the administered liposomal formulation. On the other hand, immune targeted liposomes for immunotherapy and vaccination purposes could also be developed.

The study aim was to analyze whether microvesicles and exosomes, named extracellular vesicles (EVs), purified from Toxoplasma gondii are able to stimulate the protective immunity of experimental mice when administered, as challenge, a highly virulent strain. EVs excreted from T. gondii tachyzoites (RH strain) were purified by chromatography and used for immunization assays in inbred mouse groups (EV-IM). Chronic infected (CHR) and naive (NI) mice were used as control groups, since the immune response is well known. After immunizations, experimental groups were challenged with 100 tachyzoites. Next, parasitemias were determined by real-time PCR (qPCR), and survival levels were evaluated daily. The humoral response was analyzed by detection of IgM, IgG, IgG1 and IgG2a, and opsonization experiments. The cellular response was evaluated in situ by immunohistochemistry on IFN-γ, IL-10, TNF-α and IL-17 expression in cells of five organs (brain, heart, liver, spleen and skeletal muscles). EV immunization reduced parasitemia and increased the survival index in two mouse lineages (A/Sn and BALB/c) infected with a lethal T. gondii strain. EV-IM mice had higher IgG1 levels than IgM or IgG2a. IgGs purified from sera of EV-IM mice were able to opsonize tachyzoites (RH strain), and mice that received these parasites had lower parasitemias, and mortality was delayed 48 h, compared with the same results from those receiving parasites opsonized with IgG purified from NI mice. Brain and spleen cells from EV-IM mice more highly expressed IFN-γ, IL-10 and TNF-α. In conclusion, EV-immunization was capable of inducing immune protection, eliciting high production of IgG1, IFN-γ, IL-10 and TNF-α.

Prescottella equi (formerly Rhodococcus equi) is a facultative intracellular bacterial pathogen that causes severe pneumonia in foals 1–6 months of age, whereas adult horses are highly resistant to infection. We have shown that vaccinating pregnant mares against the conserved surface polysaccharide capsule, β-1 → 6-linked poly-N-acetyl glucosamine (PNAG), elicits opsonic killing antibody that transfers via colostrum to foals and protects them against experimental infection with virulent. R. equi. We hypothesized that equine IgG1 might be more important than IgG4/7 for mediating protection against R. equi infection in foals. To test this hypothesis, we compared complement component 1 (C1) deposition and polymorphonuclear cell-mediated opsonophagocytic killing (OPK) mediated by IgG1 or IgG4/7 enriched from either PNAG hyperimmune plasma (HIP) or standard plasma. Subclasses IgG1 and IgG4/7 from PNAG HIP and standard plasma were precipitated onto a diethylaminoethyl ion exchange column, then further enriched using a protein G Sepharose column. We determined C1 deposition by enzyme-linked immunosorbent assay (ELISA) and estimated OPK by quantitative microbiologic culture. Anti-PNAG IgG1 deposited significantly (P < 0.05) more C1 onto PNAG than did IgG4/7 from PNAG HIP or subclasses IgG1 and IgG4/7 from standard plasma. In addition, IgG1 from PNAG HIP mediated significantly (P < 0.05) greater OPK than IgG4/7 from PNAG HIP or IgG1 and IgG4/7 from standard plasma. Our findings indicate that anti-PNAG IgG1 is a correlate of protection against R. equi in foals, which has important implications for understanding the immunopathogenesis of R. equi pneumonia, and as a tool for assessing vaccine efficacy and effectiveness when challenge is not feasible.

Galactoside-binding lectin (galectin) is a type of pathogen recognition molecule that occupies an important position in the invertebrate innate immunity system. Our previous study has identified a galectin gene in mollusk Solen grandis (SgGal-1) and illustrated its potential roles in innate immunity. By the functional study using recombinant protein and specific antibody, here, we confirmed the pivotal roles of SgGal-1 in immune defense of S. grandis. SgGal-1 protein was expressed in many tested tissues including gill, mantle, hepatopancreas and gonad, except hemocytes and muscle. The recombinant SgGal-1 (rSgGal-1) bound PGN and β-glucan instead of LPS in vitro, and it further caused significant agglutination of five different microbes, suggesting SgGal-1 served as a pattern recognition receptor (PRR) involved in immune defense of mollusk. Furthermore, SgGal-1 recruited hemocytes to encapsulate, which was blocked by anti-rSgGal-1 serum. In the meantime, rSgGal-1 as well as promoted the phagocytosis of hemocytes against Escherichia coli in vitro. All these results suggested that SgGal-1 in S. grandis not only acted as a PRR recognizing microbes but also directly participated in the process of immune opsonization to protect the host from pathogenic infection.

The Gram-negative bacterium Haemophilus influenzae (H. influenzae) can commensally colonize the upper respiratory tract, but also cause life threatening disease including epiglottitis, sepsis and meningitis. The H. influenzae capsule protects the bacteria against both phagocytosis and opsonization. Encapsulated H. influenzae strains are classified into serotypes ranging from a to f dependent on their distinct polysaccharide capsule. Due to the implementation of vaccination the incidence of invasive H. influenzae type b (Hib) infections has strongly decreased and infections with other capsulated types, including H. influenzae type f (Hif), are emerging. The pathogenesis of H. influenzae meningitis is not clarified. To enter the central nervous system (CNS) the bacteria generally have to cross either the blood-brain barrier (BBB) or the blood-cerebrospinal fluid barrier (BSCFB). Using a cell culture model of the BCSFB based on human choroid plexus papilloma (HIBCPP) cells and different H. influenzae strains we investigated whether Hib and Hif invade the cells, and if invasion differs between encapsulated vs. capsular-deficient and fimbriated vs. non-fimbriated variants. We find that Hib can adhere to and invade into HIBCPP cells. Invasion occurs in a strongly polar fashion, since the bacteria enter the cells preferentially from the basolateral "blood "side. Fimbriae and capsule attenuate invasion into choroid plexus (CP) epithelial cells, and capsulation can influence the bacterial distribution pattern. Finally, analysis of clinical Hib and Hif isolates confirms the detected invasive properties of H. influenzae. Our data point to roles of capsule and fimbriae during invasion of CP epithelial cells.

pH sensitives carbonate apatite (CA) has emerged as a targeted delivery vehicle for chemotherapeutics agent with tremendous potential to increase the effectivity of breast cancer treatment. The major challenge for intravenous delivery of drug-incorporated nanoparticles is their rapid opsonization, resulting in accumulation within the organs of reticuloendothelial system, such as liver and spleen. Therefore, surface modification by polyethylene glycol was implemented to improve the half-life of drug-particle complexes and enhance their uptake by target tissues. A simple, rapid, and sensitive triple quadrupole liquid chromatography–mass spectrometry method was developed and validated for quantification of gemcitabine in plasma, various organs and tumor tissues of mice with breast carcinoma, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis, quadrupole-time of flight liquid chromatography–mass spectrometry and analysis by SwissProt.Mus_musculus database were performed for protein separation, identification, and homology search by comparing the de novo sequence tag. PEGylated CA exhibited almost 6-fold increase in gemcitabine accumulation in tumor with significant reduction in other organs within 1 h of intravenous administration, compared to free drug. In addition, plasma drug amount was found to be higher in PEGylated particles, implying their role in prolonging blood circulation time of particle-bound gemcitabine. Investigation of protein corona composition demonstrated notable reduction in opsonin interactions after PEGylation of CA particles. Overall, the results indicate that the composition and dynamics of protein corona subjected to alteration by PEGylation play crucial roles in affecting successful nanoparticle-based targeted delivery of a cytotoxic drug.

Summary Objectives The opportunistic pathogen Staphylococcus epidermidis is progressively involved in device-related infections. Since these infections involve biofilm formation, antibiotics are not effective. Conversely, a vaccine can be advantageous to prevent these infections. In view of vaccine development, predicted surface proteins were evaluated on their potential as a vaccine target. Methods Immunoglobulins directed against S. epidermidis surface proteins SesB, M, O, Q and R were used to firstly affirm their surface location. Further, inhibitory effects of these IgGs on biofilm formation were determined in vitro on polystyrene and polyurethane surfaces and in vivo using a subcutaneous catheter mouse model. We also examined the opsonophagocytotic capacity of these IgGs. Results Surface localization of the five Ses proteins was demonstrated both for planktonic and sessile cells, though to a variable extent. Ses-specific IgGs added to planktonic cells had a variable inhibitory effect on cell adhesion to polystyrene, while only anti-SesO IgGs decreased cell attachment to polyurethane catheters. Although phagocytic killing was only obtained after opsonization with SesB-specific IgGs, a significant reduction of in vivo formed biofilms was observed after administration of SesB-, SesM- and SesO-specific IgGs. Conclusions Regardless of their characterization or function, S. epidermidis surface proteins can be adequate targets for vaccine development aiming the prevention of device-related infections caused by invasive S. epidermidis strains.

The complement system represents the first line of defense that is involved in the clearance of pathogens, dying cells and immune complexes via opsonization, induction of an inflammatory response and the formation of a lytic pore. Red blood cells (RBCs) are very important for the delivery of oxygen to tissues and are continuously in contact with complement proteins in the blood plasma. To prevent complement activation on RBCs, various complement regulatory proteins can be found in plasma and on the cell membrane. RBCs are special cells without a nucleus and having a slightly different make-up of complement regulators than nucleated cells, as membrane cofactor protein (MCP) is not expressed and complement receptor 1 (CR1) is highly expressed. Decreased expression and/or function of complement regulatory proteins may result in unwanted complement activation and accelerated removal of RBCs. This review describes complement regulation on RBCs and the consequences when this regulation is out of balance.

Rapid elimination of microbes from the bloodstream, along with the ability to mount an adaptive immune response, are essential for optimal host-defense. Kupffer cells are strategically positioned in the liver sinusoids and efficiently capture circulating microbes from the hepatic artery and portal vein, thus preventing bacterial dissemination. In vivo and in vitro studies have probed how complement receptor of the immunoglobulin superfamily (CRIg), also referred to as Z39Ig and V-set and Ig domain-containing 4 (VSIG4), acts as a critical player in pathogen recognition and clearance. While recent data suggested that CRIg may bind bacterial cell wall components directly, the single transmembrane receptor is best known for its interaction with complement C3 opsonization products on the microbial surface. On Kupffer cells, CRIg must capture opsonized microbes against the shear forces of the blood flow. In vivo work reveals how immune adherence (IA), a process in which blood platelets or erythrocytes associate with circulating bacteria, plays a critical role in regulating pathogen capture by CRIg under flow conditions. In addition to its typical innate immune functions, CRIg was shown to directly and indirectly influence adaptive immune responses. Here, we review our current understanding of the diverse roles of CRIg in pathogen elimination, anti-microbial immunity and autoimmunity. In particular, we will explore how, through selective capturing by CRIg, an important balance is achieved between the immunological and clearance functions of liver and spleen.

During infection and budding, human immunodeficiency virus-1 (HIV-1) acquires regulators of Complement Activation (RCAs) along with the host cell membrane on the viral envelope. Activation of host complement system results in opsonization of virus by complement fragments, however the virus evades complement mediated lysis (CoML) by virtue of the RCAs on the viral envelope. The RCAs on HIV-1 envelope process complement protein C3 into various fragments that promote viral entry and infection of cells through different complement receptors. Complement opsonized HIV-1 has been shown in vitro to infect dendritic cells (DCs) in a CR3 dependent manner, although the role of CR3 and CD46 in natural HIV-1 infection is not clear. Surface expression of CR3 and CD46 on DC subsets of 30 antiretroviral naïve, 31 treated (cART) HIV-1 infected individuals and 30 seronegative controls was measured by flow cytometry and plasma levels of cytokines and complement activity (C3c levels) were quantitated by sandwich ELISA. Significantly lower surface expression of CR3 and CD46 was observed on DC subsets in naïve and treated HIV-1 infected individuals compared to controls. Significantly higher complement activation and plasma levels of IL-4, IL-8, IL-10 and IFN-γ were observed in treatment naïve HIV-1 infected individuals than controls. Significantly lower plasma levels of IL-4, IL-6, IL-8 and IL-10 were observed in treated vs. naïve HIV-1 infected individuals. Our findings suggest that alterations in expression of CR3 and CD46 on DCs along with complement activity could be factors that influence viral persistence and HIV-1 disease progression and need to be further evaluated.

In this study, we evaluated the viability, intracellular production of reactive oxygen species (ROS) and phagocytosis of Staphylococcus aureus by blood polymorphonuclear leukocytes (PMNLs) in primiparous and pluriparous Santa Ines and Lacaune ewes. Blood was collected from 14 Santa Ines ewes (seven primiparous and seven pluriparous) and 14 Lacaune ewes (seven primiparous and seven pluriparous). The viability of PMNLs, intracellular ROS production, and S. aureus phagocytosis were evaluated using flow cytometry. No parity effect on blood’s PMNLs functionality was found. However, comparing primiparous and pluriparous ewes, we noticed a difference in the opsonized phagocytic response to S. aureus, suggesting that pluriparous ewes could be better protected against S. aureus, although further field studies are needed. Antibody-mediated opsonization of S. aureus was associated with a lower intensity of S. aureus phagocytosis in both primiparous and pluriparous ewes, while it had no effect on the percentage of S. aureus phagocytosis. The percentage and geometric mean fluorescence intensity (GMFI) of S. aureus phagocytosis behaved differently in Lacaune and Santa Ines ewes, which could be explained by breed and farm effects. The intensity of intracellular ROS production by PMNLs when stimulated with S. aureus was higher than that of unstimulated basal PMNLs in both primiparous and pluriparous ewes, while no effect on the percentage of PMNLs that produced ROS was observed. Thus, our study provides novel insights into PMNLs’ functionality in primiparous and pluriparous ewes, which could impact their health, although further research is needed to extrapolate conclusions to the field.

Dexmedetomidine (DEX) is a highly selective α2-adrenoceptor agonist, which can regulate inflammatory responses. However, whether DEX interferes with the inflammation resolving remains unclear. Here, we reported the effects of DEX on zymosan-induced generalized inflammation in mice during resolution. Mice were administered intraperitoneally with DEX after the initiation of sepsis. The resolution interval (Ri), a vital resolution indice, decreased from twelve hours to eight hours after the administration of DEX. The induction of peritoneal pro-inflammatory interleukin [IL] - 1β and tumour necrosis factor-α (TNF-α) appeared to be inhibited. Of interest, the anti-inflammatory transforming growth factor-β1 (TGF-β1) but not IL-10 levels were up-regulated at twenty-four hours in the DEX group along with 1.0 mg/mice zymosan A (ZyA) treatment. The expression levels of multiple genes related to protective immune processes and clearance functions were detected and revealed the same trends. DEX markedly increased the F4/80+Ly6G+ macrophage population. Additionally, the adequate apoptotic neutrophil clearance from injury after DEX installation could be reverse by opsonization or co-instillation of TGF-β1 neutralizing antibody in vivo, promoting the inflammation-resolution programs. In conclusion, DEX post-treatment, via the increase of F4/80+Ly6G+ macrophages, provokes further secretion of TGF-β1, leading to the attenuated cytokine storm and accelerated inflammation resolving.

Two clinical tests - the erythrocyte sedimentation rate and the opsonic index - have long been known to non-specifically detect pathology based on their responsiveness to changes in serum proteins. In infections serum levels of specific antibodies increase. However, for healthy subjects Wright held that antibodies contributed minimally to opsonic activity (the complement-enhanced phagocytosis of microorganisms). The activity was present in newborn serum, was increased in the acute phase of an immune response prior to antibody increase, and was less specific. Furthermore, defective opsonization was associated with undue susceptibility to certain infections, for which a genetic basis was later found. With the demonstrations of complement-mediated lysis both of normal cells by foreign (plant) lectins, and of foreign cells (microorganisms) by animal lectins, it now appears that endogenous lectins correspond to the heat-stable component of Wright's serum opsonic activity. His work leads to the lectin pathway of complement activation with specificities limited to the recognition of relatively immutable surface sugars - predictable pathogen characters that contrast with the less predictable targets of the adaptive immune system.

Ficolins are pattern-recognition molecules (PRMs) that could form complexes with mannose-binding lectin-associated serine proteases (MASPs) to trigger complement activation via the lectin pathway, thereby mediating a series of immune responses including opsonization, phagocytosis and cytokine production. In the past few decades, accumulating evidence have suggested that ficolins play a major role in the onset and development of several autoimmune diseases (ADs), including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), Type 1 diabetes (T1D), inflammatory bowel disease (IBD), etc. In this review, we synthesized previous literatures and recent advances to elucidate the immunological regulations of ficolins and discuss the potential diagnostic ability of ficolins in ADs, as well as giving an insight into the future therapeutic options for ficolins in ADs.

Surfactant Protein D (SP-D) is a collectin protein that participates in the innate immune defense of the lungs. SP-D mediates the clearance of invading microorganisms by opsonization, aggregation or direct killing, which are lately removed by macrophages. SP-D is found as a mixture of trimers, hexamers, dodecamers and higher order oligomers, “fuzzy balls”. However, it is unknown whether there are differences between these oligomeric forms in functions, activity or potency. In the present work, we have obtained fractions enriched in trimers, hexamers and fuzzy balls of full-length recombinant human (rh) SP-D by size exclusion chromatography, in a sufficient amount to perform functional assays. We have evaluated the differences in protein lectin-dependent activity relative to aggregation and binding to E. coli, one of the ligands of SP-D in vivo. Fuzzy balls are the most active oligomeric form in terms of binding and aggregation of bacteria, achieving 2-fold binding higher than hexamers and 50% bacteria aggregation at very short times. Hexamers, recently described as a defined oligomeric form of the protein, have never been isolated or tested in terms of protein activity. rhSP-D hexamers efficiently bind and aggregate bacteria, achieving 50–60% aggregation at final time point and high protein concentrations. Nevertheless, trimers are not able to aggregate bacteria, although they bind to them. Therefore, SP-D potency, in functions that relay on the C-lectin activity of the protein, is proportional to the oligomeric state of the protein., This work has been supported by grants from the Spanish Ministry of Science and Innovation (RTI2018-094564-B-I00 to J.P.-G) and the Regional Government of Madrid (P2018/NMT­4389 to J.P.-G.) F.M.-H. Acknowledges support from the Spanish Ministry of Science Innovation and Universities (project BFU2017-83794-P, (AEI/FEDER, UE)) and from the European Research Council (ERC) under the European Union Horizon 2020 research and innovation (grant agreement No 681299). R.A. acknowledges an FPU PhD fellowship from the Spanish Ministry of Education.

Phagocytosis is an evolutionarily conserved immune response, whose efficiency is fundamentally coupled with opsonization of extracellular microbes. How marine mollusks cells recognize and selectively capture pathogens during phagocytosis to clear them is not completely understood. In this study, we observed that plasma is extremely effective for oyster hemocyte phagocytosis, so we investigated candidate proteins among plasma proteins with binding affinity for Vibrio parahaemolyticus in Pacific oyster (Crassostrea gigas) by subjecting them to mass spectroscopy analysis for protein identification and characterization, and address the complex regulatory network to engulf invaders. There were 620 identified proteins potentially associated with bacteria binding and phagocytosis which could be quantified. Our results showed that C1q and lectins identified in Pacific oyster plasma held binding ability to bacteria, clearly suggesting their potent to be opsonins. The dominant expressed plasma protein p1-CgC1q (Complement component 1q)-like protein was identified and its opsonic role was confirmed in this study. The cell surface receptor Cgintegrin interacts directly with p1-CgC1q to mediate phagocytosis. We further confirmed that the interaction between C1q and integrin not rely on the typical recognition site RGD but on the RGE. Evidence exist revealed that p1-CgC1q could coat bacteria via the endotoxin LPS (lipopolysaccharide) and subsequently bind the receptor integrin to significantly enhance hemocytic phagocytosis and bacteria clearance. This study has thus furnished clear evidence for the importance of plasma proteins in mollusk, shedding light on the humoral immunity and an underappreciated strategy in marine host-pathogen interactions.

Some serovars of salmonella cause huge global diseases such as enteric fever and invasive non typhoidal Salmonella disease. Flagellin as a key antigenic component of salmonella, can induce humoral and cellular immunity responses. In this research, we performed an opsonophagocytic killing assay (OPKA) as an important mechanism of the host-defense system, for salmonella to study the activity of anti-sera of native FliC, truncated modified recombinant FliC (tmFliC) and full length recombinant FliC proteins (flFliC). Also, the potency of antibodies for inhibiting bacterial movement was evaluated by traditional and newly-designed motility inhibition assay methods. Results showed both recombinant FliC anti-sera and native FliC (nFliC) anti-serum had the ability to opsonize Salmonella typhimurim, which led to bacterial clearance by mice macrophages. Also, inhibition of bacterial motility was observed for all anti-sera. Anti-nFliC and anti-flFliC sera showed higher effects on Salmonella typhimurim motility than that of tmFliC. In traditional method, about 88%, 86% and 80% inhibition were observed by using 5% nFliC, anti-flFliC and anti-tmFliC sera, respectively. In the newly-designed method using SIM (Sulfide indole motility) medium, results confirmed the traditional method for motility inhibition. Our findings suggest that salmonella fliC as a protective antigen may disrupt the flagellum apparatus activity.

C-type lectins are a superfamily of Ca2+-dependent carbohydrate-recognition proteins that function as pattern recognition receptors (PRRs) in innate immune system. In this study, a new C-type lectin was identified from the swimming crab Portunus trituberculatus (PtCLec1). The full-length cDNA of PtCLec1 was 873 bp encoding 176 amino acids. The predicted PtCLec1 protein contained a signal peptide and a single carbohydrate-recognition domain with a special YPD motif. The PtCLec1 transcripts were mainly detected in hepatopancreas and its relative expression levels were significantly up-regulated after the challenges of Vibrio alginolyticus, Micrococcus luteus and Pichia pastoris. The recombinant PtCLec1 (rPtCLec1) could bind all the tested pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides (LPS), peptidoglycan (PGN) and glucan (GLU), and microorganisms, including V. alginolyticus, V. parahaemolyticus, Pseudomonas aeruginosa, Staphylococcus aureus, M. luteus and P. pastoris. It also exhibited strong activity to agglutinate bacteria and yeast in a Ca2+-dependent manner, and such agglutinating activity could be inhibited by d-galactose and LPS. Moreover, rPtCLec1 revealed antimicrobial activity against the tested Gram-negative (V. alginolyticus, V. parahaemolyticus and P. aeruginosa) and Gram-positive bacteria (S. aureus and M. luteus), and promoted the clearance of V. alginolyticus in vivo and hemocyte phagocytosis in vitro. Knockdown of PtCLec1 could down-regulate the expression of phagocytosis-related genes, but enhance the expression levels of prophenoloxidase (proPO) system-related genes, mannose-binding lectin (MBL), antimicrobial peptides (AMPs), MyD88 and Relish. All these results indicate that PtCLec1 might act as a PRR in immune recognition and an opsonin in pathogen elimination.

No studies showed specific antibody levels against all serotypes covered by 13-valent pneumococcal conjugate vaccine (PCV13) among polyclonal intravenous immunoglobulin (IVIG) products. Our study aimed to assess whether we could expect the efficacy of IVIG therapy for invasive pneumococcal disease (IPD) and to clarify the age group which should be recommended for IVIG therapy in case of IPD. Serotype-specific immunoglobulin G (IgG) levels against PCV13 serotypes were measured in four IVIGs which were produced from Japanese donors who were not immunized with any pneumococcal conjugate vaccines (PCVs), and in the serum of 160 non-PCV immunized Japanese subjects, by enzyme-linked immunosorbent assay. The functional opsonic activities of the IVIGs against serotypes 6B and 19A were assessed by a multiplexed opsonophagocytic killing assay. Japanese infants aged

Background Collectins are considered to play a role in host defense via complement activation and opsonization, and are composed of a collagen-like domain and a carbohydrate recognition domain (CRD). Collectin placenta 1 (CL-P1) showed scavenger receptor activity as functions in vitro, and has three candidate domains: a coiled-coil domain, a collagen-like domain and CRD. Methods We constructed seven types of CL-P1 deletion mutants to determine the site of each ligand binding domain, and observed whether the specific binding to sugar ligand, microbes, or oxidized LDL decreases or not in cells with CL-P1 deletion mutants and CL-P1 containing mutations of amino acid, respectively. Results CL-P1 mainly interacted with ligands of microbes through the collagen-like domain and it binds a sugar ligand through the CRD. Additionally it could bind oxidized low density lipoprotein (OxLDL) due to the coiled-coil domain as well as the collagen-like domain. This binding study using mutants at three positively charged sites in the collagen-like domain reveals that the site of R496 K499 K502 plays the most important role in ligand binding functions for microbes and OxLDL. Conclusions CL-P1 has three unique functional domains: the collagen-like domain mainly acts against most negatively charged ligands, and the CRD specifically does against sugar substances, while the coiled-coil domain additionally acts on modified LDL. General significance We considered that the binding activity for various ligands due to the association of a coiled-coil domain, a collagen-like domain and/or a CRD in CL-P1, might play a role in physiological functions in the animal body.

Acute pain remains a tremendous clinical and economic burden, as its prevalence and common narcotic-based treatments are associated with poorer outcomes and higher costs. Multimodal analgesia portends great therapeutic promise, but rarely allows opioid sparing, and new alternatives are necessary. Microparticles (MPs) composed of biodegradable polymers [e.g., poly(lactic-co-glycolic acid) or PLGA] have been applied for controlled drug release and acute pain treatment research. However, foreign particles' presence within inflamed tissue may affect the drug release or targeting, and/or cause a secondary inflammatory reaction. We examined how small alterations in the particulate nature of MPs affect both their uptake into and subsequent activation of macrophages. MPs composed of PLGA and chitosan (PLGA-Chi) loaded with bupivacaine (BP) were engineered at different sizes and their opsonization by J774 macrophages was assessed. Uptake of PLGA-Chi by macrophages was found to be size dependent, but they were not cytotoxic or proinflammatory in effect. Moreover, encapsulation of MPs in a thermoresponsive loading gel (pluronic F-127) effectively prevented opsonization. Finally, MPs displayed sustained, tunable release of BP up to 7 days. These results demonstrate our ability to develop a drug delivery system capable of controlled release of local anesthetics to treat acute/subacute pain while concurrently avoiding enhanced inflammation.

The lectin pathway involves recognition of pathogen-associated molecular patterns by mannose-binding lectin (MBL), and the subsequent activation of associated enzymes, termed MBL-associated serine proteases (MASPs). In this study, we demonstrate that the transcript of MBL gene is present in the early embryo of zebrafish, and MBL protein is also present in the embryo. In addition, we show that recombinant zebrafish MBL was able to bind the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Staphylococcus aureus, and rMBL was able to promote the phagocytosis of E. coli and S. aureus by macrophages, indicating that like mammalian MBL, zebrafish MBL performs a dual function in both pattern recognition and opsonization. Importantly, we show that microinjection of anti-MBL antibody into the early developing embryos resulted in a significantly increased mortality in the embryos challenged with Aeromonas hydrophila (pathogenic to zebrafish); and injection of rMBL into the embryos (resulting in increase in MBL in the embryo) markedly promoted their resistance to A. hydrophila; and this promoted bacterial resistance was significantly reduced by the co-injection of anti-MBL antibody with rMBL but not by the injection of anti-actin antibody with rMBL. These suggest that the lectin pathway may be already functional in the early embryos in zebrafish before their immune system is fully matured, protecting the developing embryos from microbial infection. This work provides a new angle to understand the immune role of the lectin pathway in early development of animals.

Complement is usually seen to largely originate from the liver to accomplish its tasks systemically – its return to the production site has long been underestimated. Recent progress in genomics, therapeutic effects on complement, standardised possibilities in medical laboratory tests and involvement of complosome brings the complement system with its three major functions of opsonization, cytolysis and phagocytosis back to liver biology and pathology. The LOINC™ system features 20 entries for the C3 component of complement to anticipate the application of artificial intelligence data banks algorythms of which are fed with patient-specific data connected to standard lab assays for liver function. These advancements now lead to increased vigilance by clinicians. This reassessment article will further elucidate the distribution of synthesis sites to the three germ layer-derived cell systems and the role complement now known to play in embryogenesis, senescence, allotransplantation and autoimmune disease. This establishes the liver as part of the gastro-intestinal system in connection with nosological entities never thought of, such as the microbiota-liver-brain axis. In neurological disease etiology infectious and autoimmune hepatitis play an important role in the context of causative viz reactive complement activation. The mosaic of autoimmunity, i.e. multiple combinations of the many factors producing varying clinical pictures, leads to the manifold facets of liver autoimmunity., Graphical abstract Image 1, Highlights • Liver pathology associated to complement system derangement. • Liver autoimmunity depends on complement system activation. • The complement system may turn itself against its production site, i.e. the hepatocyte. • Complosome, i.e. intracellular metabolic involvement of complement components, scrutinized in liver pathology.

An observational study was conducted to determine immunogenicity before and after primary and secondary vaccinations with 23-valent pneumococcal polysaccharide vaccine in a cohort of 40 elderly patients with chronic lung diseases. Safety of this vaccine was also compared between primary and secondary vaccination. We analyzed serotype-specific immunoglobulin G (IgG) and the opsonization index (OI) for serotypes 6B, 14, 19F, and 23F and compared adverse local and systemic reactions. The levels of serotype-specific IgG and the OIs significantly increased 1 month after primary and secondary vaccinations. Peak levels of IgG after secondary vaccination were 5-20% lower than those after primary vaccination, while serotype-specific OIs after secondary vaccination were comparable with those after primary vaccination. The levels of serotype-specific IgG required for 50% killing significantly decreased 1 month after vaccination. These values for serotypes 14, 19F, and 23F were slightly elevated immediately before secondary vaccination, but those for serotype 6B did not change. After secondary vaccination, these values declined slightly for serotypes 14, 19F, and 23F and remained low for serotype 6B. Although self-limited local and systemic reactions were more frequent after secondary vaccination compared with primary vaccination, no serious systemic reaction was found after either vaccination. Our data suggest a sustained functional serotype-specific IgG after primary and secondary vaccination and confirmed the safety of secondary vaccination among elderly individuals with chronic lung disease.

Group A Streptococcus pyogenes (GAS) strain AP53 is a primary isolate from a patient with necrotizing fasciitis. These AP53 cells contain an inactivating mutation in the sensor component of the cluster of virulence (cov) responder (R)/sensor (S) two-component gene regulatory system (covRS), which enhances the virulence of the primary strain, AP53/covR+S−. However, specific mechanisms by which the covRS system regulates the survival of GAS in humans are incomplete. Here, we show a key role for covRS in the regulation of opsonophagocytosis of AP53 by human neutrophils. AP53/covR+S− cells displayed potent binding of host complement inhibitors of C3 convertase, viz. Factor H (FH) and C4-binding protein (C4BP), which concomitantly led to minimal C3b deposition on AP53 cells, further showing that these plasma protein inhibitors are active on GAS cells. This resulted in weak killing of the bacteria by human neutrophils and a corresponding high death rate of mice after injection of these cells. After targeted allelic alteration of covS− to wild-type covS (covS+), a dramatic loss of FH and C4BP binding to the AP53/covR+S+ cells was observed. This resulted in elevated C3b deposition on AP53/covR+S+ cells, a high level of opsonophagocytosis by human neutrophils, and a very low death rate of mice infected with AP53/covR+S+. We show that covRS is a critical transcriptional regulator of genes directing AP53 killing by neutrophils and regulates the levels of the receptors for FH and C4BP, which we identify as the products of the fba and enn genes, respectively. Background: Host complement-mediated opsonophagocytosis is an innate immune response to GAS. Results: Inactivation of the covS component of the covRS system allows GAS to avoid opsonization. Conclusion: Bacterial genes that subvert opsonization of GAS are regulated by the covRS system. Significance: The covRS system regulates complement inhibitor binding to GAS via the products of the fba and enn genes.

C-type lectins are a superfamily of Ca2+-dependent carbohydrate-recognition proteins consisting of at least one carbohydrate-recognition domain (CRD), which participate in nonself-recognition and clearance of invaders. In invertebrate, some multidomain C-type lectins have been identified, but their relative functions and binding mechanism are still meager. In the present study, A C-type lectin (CfLec-4) with four CRDs from Chlamys farreri was selected to investigate its possible function in innate immunity. The mRNA expression of CfLec-4 in hemocytes was significantly up-regulated (P < 0.01) after the stimulations of beta-glucan, LPS or PGN, and reached the highest expression level at 3, 6, 12 h post-stimulation, which was 27.9-, 22.6- or 47.9-fold of that in blank group, respectively. Immunohistochemistry assay with polyclonal antibody specific for CfLec-4 revealed that the endogenous CfLec-4 was mainly located in the hepatopancreas, kidney and gonad of the scallops. The recombinant CfLec-4 (rCflec-4) could bind LPS, PGN, glucan and mannose in vitro, but could not bind LTA. Furthermore, rCflec-4 displayed a broader bacteria binding spectrum towards Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus as well as Gram-negative bacteria Escherichia coil, Vibrio anguillarum and fungi Pichia pastoris. Meanwhile, rCfLec-4 could significantly (P < 0.01) enhance the phagocytosis of hemocytes in vitro. The results clearly suggested that four-CRD containing CfLec-4 not only served as PRR with wider recognition spectrum, but also functioned as an opsonin participating in the clearance of invaders in scallops. It could be inferred that the diversity and complexity of CRDs in C-type lectins endowed these receptors with comprehensive recognition spectrum and multiple immune functions against complex living environment. (C) 2012 Elsevier Ltd. All rights reserved.

Basic studies have proven that cranberries may prevent urinary tract infections through changing the adhesiveness of Escherichia coli (E. coli) to urothelial cells. Various cranberry preparations, including extract powder, capsules, and juice, have been shown to be effective in clinical and epidemiological research. Because cranberries are most commonly consumed as juice in a diluted concentration, the aim of this study was to investigate whether the equivalent daily dose of cranberry juice is sufficient to modify host urine to change the uropathogenicity of E. coli. Urine from rats taking an equivalent daily dose of cranberry juice has been shown to decrease the capability of E. coli in hemagglutination, urothelium adhesion, nematode killing, and biofilm formation. All these changes occurred after E. coli was incubated in cranberry metabolite-containing urine, defined as urine opsonization. Urine opsonization of E. coli resulted in 40.9 % (p = 0.0038) decrease in hemagglutination ability, 66.7 % (p = 0.0181) decrease in urothelium adhesiveness, 16.7 % (p = 0.0004) increase in the 50 % lethal time in killing nematodes, and 53.9 % (p = 5.9 × 10−4) decrease in biofilm formation. Thus, an equivalent daily dose of cranberry juice should be considered sufficiently potent to demonstrate urine opsonization in E. coli.

Microbial resistance to antimicrobial drugs is promoting a search for new antimicrobial agents that target highly conservative structures of pathogens. Human CD14 – a known pattern recognition receptor (PRR) which recognizes multiple ligands from different microbes might be a worthy candidate. The aim of our work was to create a CD14/Fc dimer protein and evaluate its whole bacteria binding and opsonizing capabilities. Fusion of CD14 with the fragment crystallisable (Fc) part of human IgG1 could not only lead to an artificial opsonin but the dimerization through the Fc part might also increase its affinity to different ligands. Human CD14 and the Fc part of human IgG1 was fused and expressed in HEK293 cells. A histidine tagged CD14 (CD14/His) was also expressed as control. Using flow cytometry we could prove that CD14/Fc bound to whole Gram-negative bacteria, especially to short lipopolysaccharide (Ra and Re) mutants, and weak interaction was observed between the fusion protein and Listeria monocytogenes . Other Gram-positive bacteria and fungi did not show any association with CD14/Fc. CD14/His showed about 50-times less potent binding to Gram-negative bacteria. CD14/Fc acted as an opsonin and enhanced phagocytosis of these bacteria by neutrophil granulocytes, monocyte-derived macrophages and dendritic cells. Internalization of bacteria was confirmed by trypan blue quenching and confocal microscopy. On neutrophils the Fc part of the fusion protein was recognized by Fc receptors (CD16, CD32), as determined by blocking experiments. CD14/Fc enhanced the killing of bacteria in an ex vivo whole blood assay. Our experiments confirm that PRR/Fc fusion proteins can give a boost to FcR dependent phagocytosis and killing provided the antimicrobial part binds efficiently to microbes.

Leptospirosis is a widespread re-emerging zoonosis of human and veterinary concern. It has been shown that virulent leptospires protect themselves against the host’s innate immune system, a strategy that allows the bacteria to reach immunologically safe environments. Although extensive studies on host–pathogen interactions have been performed, little is known on how leptospires deal with host immune attack. In a previous work, we demonstrated the ability of leptospires to bind human plasminogen (PLG), that after treatment with activators, conferred plasmin (PLA) activity on the bacteria surface. In this study, we show that the PLA activity associated to the outer surface of Leptospira could interfere with the host immune attack by conferring some evasion advantage during infection. We demonstrate that PLA-coated leptospires interfere with complement C3b and IgG depositions on the bacterial surface, probably through the degradation of these components, thus diminishing opsonization process. Similar decrease on the deposition was observed when normal and immune sera from patients diagnosed with leptospirosis were employed as a source of IgG. We believe that decreasing opsonization by PLA generation might be an important aspect of the leptospiral immune escape strategy and survival. To our knowledge, this is the first proteolytic activity of plasmin associated-Leptospira related to anti-opsonic properties reported to date.

Vitellogenin (Vg), once reported to be a female-specific protein, has been identified in both male and juvenile fishes. However, the biological significance of the production of Vg in the male and juvenile fishes is elusive. Our previous studies showed that Vg is an opsonin capable of enhancing phagocytosis, but the mechanism by which Vg mediates phagocytosis is unknown. In this study we demonstrated that Vg-opsonized phagocytosis was characterized by pseudopod extension and depended upon tyrosine kinase. In contrast, inhibition of Rho family proteins and microtubule depolymerization had little effects on Vg-opsonized phagocytosis. Besides, Vg-opsonized phagocytosis was substantially blocked by monoclonal antibodies against FcγRs but not by CR3 antibody. Moreover, theoretical prediction analysis further revealed that Vg had the potency to interact with Fcγ receptors. Finally, the expression of proinflammatory cytokine genes tnf-α and il-1β was significantly up-regulated by Vg, and this up-regulation was inhibited by selective inhibitors of FcR signaling pathways, wortmannin and piceatannol. Taken together, these results suggest that Vg plays an IgG-like role in that it activates FcγR-mediated phagocytosis, thus establishing an antibody-like function for Vg for the first time.

The initial interaction of Gram-negative bacteria with erythrocytes and its implications on leukocyte phagocytosis and oxidative burst in human whole blood were examined. Alexa-labeled Escherichia coli, wild-type H44/76 N. meningitidis and the H44/76lpxA lipopolysaccharide (LPS)-deficient mutant were incubated with whole blood using lepirudin as anticoagulant which has no adverse effects on complement. Bacteria free in plasma, bound to erythrocytes or phagocytized by granulocytes and monocytes were quantified using flow cytometry. The effects of the C3 inhibitor compstatin, a C5a receptor antagonist (C5aRa) and a complement receptor 1 (CR1)-blocking antibody (3D9) were examined. Most bacteria (80%) immediately bound to erythrocytes. The binding gradually declined over time, with a parallel increase in phagocytosis. Complement inhibition with compstatin reduced erythrocyte binding and bacterial C3 opsonization. In contrast, the C5aRa efficiently reduced phagocytosis, but did not affect the binding of bacteria to erythrocytes. The anti-CR1 blocking mAb dose-dependently reduced bacterial binding to erythrocytes to nil, with subsequent increased phagocytosis and oxidative burst. LPS had no effect on these processes since similar results were obtained using an LPS-deficient N. meningitidis mutant. In vivo experiments in a pig model of sepsis showed limited binding of bacteria to erythrocytes, consistent with the facts that erythrocyte CR1 receptors are absent in non-primates and that the bacteria were mainly found in the lungs. In conclusion, complement-dependent binding of Gram-negative bacteria to erythrocyte CR1 decreases phagocytosis and oxidative burst by leukocytes in human whole blood. (C) 2011 Elsevier Ltd. All rights reserved.

Hospital-acquired infections (HAIs) remain a leading cause of death in the United States. Unfortunately, treatment of HAIs is complicated by the emergence of antibiotic-resistant bacterial strains. In an effort to enhance the body’s natural immune response to infection, we have developed an artificial opsonin to promote the recognition, phagocytosis, and destruction of pathogenic bacteria by human phagocytes. The artificial opsonin is constructed from multivalent conjugates of poly( l -lysine)-graft-poly(ethylene glycol) with vancomycin and human IgG-Fc. Our approach utilizes vancomycin’s inherent ability to bind to d -Ala- d -Ala terminated peptides present in the cell wall of Gram-positive bacteria. Here, we show that conjugation of vancomycin to PLL-g-PEG prevents its action as an antibiotic and allows vancomycin to function solely as a recognition molecule. Human IgG-Fc antibody fragment serves as a phagocyte recognition molecule and is recognized by the Fcγ cell surface receptors expressed on professional human phagocytes. Using flow cytometry, we found that a polysaccharide-encapsulated, methicillin-resistant strain of Staphylococcus epidermidis is efficiently recognized by the artificial opsonin (nearly 100% of cells were opsonized) and that opsonin binding is specific since it can be inhibited by the soluble cell wall peptide analog acetyl-Lys- d -Ala- d -Ala. Opsonization of S. epidermidis resulted in an approximate 2-fold increase in phagocytosis by a human neutrophil cell line. Notably, Enterococcus faecalis VanB, a bacterial strain with inducible vancomycin resistance, was used to show that the artificial opsonin does not unintentionally induce antibiotic resistance mechanisms.

The objective was to examine the effects of caffeine, dibutyryl cyclic AMP, and heparin on the chemotaxis and/or phagocytosis of PMNs for porcine sperm. The chemotactic activity of PMNs, determined in a blind well chamber, increased (P < 0.05) when fresh serum was added to the medium (control containing BSA, 1109.5 cells/mm(2) vs serum, 1226.3 cells/mm(2)), regardless of the presence of sperm (control, 1121.1 cells/mm(2) vs serum, 1245.8 cells/mm(2)), whereas heat-inactivated serum did not affect activity (without sperm, 1099.4 cells/mm(2) and with sperm, 1132.6 cells/mm(2)). Regardless of live and dead sperm and of the origin of PMNs (boars vs sows), the phagocytotic activity of PMNs, as determined by co-culture of PMNs with sperm for 60 min, increased (P < 0.05) in the presence of fresh serum containing active complement (46.7 and 43.0%, respectively), but stimulation was decreased (P < 0.05) when 1 mM or higher concentrations of caffeine was added to the medium (from 40.7 to 20.8-31.6%). The origin of PMNs (sows vs boars) did not significantly affect phagocytotic activity. The percentage of PMNs that phagocytized polystyrene latex beads decreased when 2 mM caffeine was added to the medium containing porcine serum (from 43.7 to 21.5%). Serum-stimulated chemotactic activity of PMNs (1089.9 cells/mm(2)) was also reduced (P < 0.05) with 2 mM caffeine (942.5 cells/mm(2)). Furthermore, dibutyryl cAMP at ≥ 0.1 mM or heparin at ≥ 100 μg/mL decreased phagocytotic activity, in a concentration-dependent manner (P < 0.05). Supplementation of PMNs with heparin at 100 or 500 μg/mL decreased (P < 0.05) chemotactic activity in the presence of serum (from 1137.1 cells/mm(2) to 1008.8-1026.3 cells/mm(2)). We inferred that opsonization in the presence of active complement stimulated phagocytotic and chemotactic activities of PMNs, whereas supplementation with caffeine and dibutyryl cAMP (which could be associated with the intracellular cAMP level of PMNs) or adding heparin decreased serum-stimulated phagocytotic and chemotactic activities.