Quasi-opsonin conjugated lipase-sensitive micelles activate macrophages against facultative intracellular bacterial infection.

Drug resistance caused by facultative intracellular bacteria such as Salmonella typhimurium (S. typhimurium) is still a tough challenge. Bacteria phagocytosed by macrophages have evolved a variety of mechanisms to defend against host attack, and the poor entry of antibiotics into infected macrophages is conducive to the survival of intracellular bacteria. In this report, we prepared a quasi-opsonized chloramphenicol (Chl)-loaded micellar system (B-mLBP-M/Chl) assembled by a bacterial lipase-sensitive polymer with a conjugate of lipopolysaccharide-binding protein (LBP) analog and biotin (B) as a ligand, which could eliminate drug-resistant S. typhimurium with quasi-opsonization via 3 steps: (i) target and release antibiotics on bacteria lipase, (ii) opsonize S. typhimurium to be digested by the macrophage, and (iii) activate the macrophage for fighting. The B-mLBP-M/Chl could target bacterial LPS through mLBP by simulating the N-terminal sequence of native LBP, exhibiting a high ability to target the localized infection site in mice. It could also activate the phagocytosis of macrophages via coupled biotin, cooperating with antibiotics and effectively improving the survival of mice with little pathological damage to tissues. Moreover, compared with native opsonin, B-mLBP does not cause an excessive inflammatory response and could recover homeostasis after exerting the quasi-opsonization by regulating the levels of pro-inflammatory cytokines and anti-inflammatory cytokines. With a universal target site for Gram-negative bacteria and macrophage activation, this B-mLBP-M/Chl could be applied to other bacterial infections in the future. In particular, this analog may also serve as a useful template to design safe artificial opsonin, which could be a ligand for drug delivery systems or prodrugs.

Characterization of the Dual Functions of LvCrustinVII from Litopenaeus vannamei as Antimicrobial Peptide and Opsonin.

Crustin are a family of antimicrobial peptides that play an important role in protecting against pathogens infection in the innate immune system of crustaceans. Previously, we identified several novel types of crustins, including type VI and type VII crustins. However, their immune functions were still unclear. In the present study, the immune function of type VII crustin LvCrustinVII were investigated in Litopenaeus vannamei. LvCrustinVII was wildly expressed in all tested tissues, with relatively high expression levels in hepatopancreas, epidermis and lymphoid organ. Upon Vibrio parahaemolyticus infection, LvCrustinVII was significantly upregulated in hepatopancreas. Recombinant LvCrustinVII (rLvCrustinVII) showed strong inhibitory activities against Gram-negative bacteria Vibrio harveyi and V. parahaemolyticus, while weak activities against the Gram-positive bacteria Staphylococcus aureus. Binding assay showed that rLvCrustinVII could bind strongly to V. harveyi and V. parahaemolyticus, as well as the cell wall components Glu, LPS and PGN. In the presence of Ca2+, rLvCrustinVII could agglutinate V. parahaemolyticus and enhance hemocyte phagocytosis. The present data partially illustrate the immune function of LvCrustinVII, which enrich our understanding on the functional mechanisms of crustins and provide useful information for application of this kind of antimicrobial peptides.

Differential Interactions of Serum and Bronchoalveolar Lavage Fluid Complement Proteins with Conidia of Airborne Fungal Pathogen Aspergillus fumigatus.

Even though both cellular and humoral immunities contribute to host defense, the role played by humoral immunity against the airborne opportunistic fungal pathogen Aspergillus fumigatus has been underexplored. In this study, we aimed at deciphering the role of the complement system, the major humoral immune component, against A. fumigatus Mass spectrometry analysis of the proteins extracted from A. fumigatus conidial (asexual spores and infective propagules) surfaces opsonized with human serum indicated that C3 is the major complement protein involved. Flow cytometry and immunolabeling assays further confirmed C3b (activated C3) deposition on the conidial surfaces. Assays using cell wall components of conidia indicated that the hydrophobin RodAp, ß-(1,3)-glucan (BG) and galactomannan (GM) could efficiently activate C3. Using complement component-depleted sera, we showed that while RodAp activates C3 by the alternative pathway, BG and GM partially follow the classical and lectin pathways, respectively. Opsonization facilitated conidial aggregation and phagocytosis, and complement receptor (CR3 and CR4) blockage on phagocytes significantly inhibited phagocytosis, indicating that the complement system exerts a protective role against conidia by opsonizing them and facilitating their phagocytosis mainly through complement receptors. Conidial opsonization with human bronchoalveolar lavage fluid (BALF) confirmed C3 to be the major complement protein interacting with conidia. Nevertheless, complement C2 and mannose-binding lectin (MBL), the classical and lectin pathway components, respectively, were not identified, indicating that BALF activates the alternative pathway on the conidial surface. Moreover, the cytokine profiles were different upon stimulation of phagocytes with serum- and BALF-opsonized conidia, highlighting the importance of studying interaction of conidia with complement proteins in their biological niche.

Rapid and Efficient Purification of Functional Collectin-12 and Its Opsonic Activity against Fungal Pathogens.

Collectin-12 (collectin placenta 1, CL-P1, or CL-12) is a newly identified pattern recognition molecule of the innate immune system. Recent evidences show that CL-12 plays important roles not only in innate immune protection against certain clinically important pathogens but also in scavenging of host molecules, leukocyte recruitment, and cancer metastasis. Furthermore, CL-12 has been shown to be associated with the pathogenesis of human diseases such as Alzheimer's disease and multiple sclerosis lesion development. Therefore, the functional consequence of CL-12 remains intriguing and awaits further elucidation. However, available protocols for the purification of recombinant CL-12 with high purity are laborious and inefficient and hamper further functional studies. Here, we report a simple, rapid, and efficient solution to obtain biologically active CL-12 with high purity. We established stable transfected Flp-In™-CHO cells expressing the recombinant CL-12 extracellular domain in high amounts. Recombinant CL-12 was purified from cell culture supernatants using a 3-step rapid purification procedure utilizing disposable affinity and ion exchange minicolumns. Purified recombinant CL-12 adopted an oligomeric structure with monomers, dimers, and trimers and retained its binding capacity towards the A. fumigatus strain that has been described before. Furthermore, we demonstrated the opsonic properties towards eight clinical isolates of A. fumigatus strains and diverse clinically important fungal pathogens. Purified recombinant CL-12 revealed a differential binding capacity towards selected fungal pathogens in vitro. In conclusion, we demonstrate a rapid and efficient purification solution for further biochemical and functional characterization of CL-12 and reveal opsonic properties of CL-12 towards diverse fungal pathogens.

A sialic acid-binding lectin with bactericidal and opsonic activities from Ruditapes philippinarum.

In the present study, a sialic acid-binding lectin was cloned and characterized from Manila clam Ruditapes philippinarum (designed as RpSabl). The open reading frame of RpSabl encoded a polypeptide of 162 amino acids with a calculated molecular mass of 17.7 kDa. Analysis of the conserved domain suggested that RpSabl was a new member of the sialic acid-binding lectins family. In non-stimulated clams, RpSabl transcripts were constitutively expressed in all five tested tissues, especially in hepatopancreas. After Vibrio anguillarum challenge, the expression of RpSabl mRNA in hepatopancreas was significantly up-regulated at 3 h (3.8-fold, P < 0.05), 6 h (4.9-fold, P < 0.05), 12 h (12.3-fold, P < 0.01) and 24 h (9.7-fold, P < 0.01), while RpSabl transcripts in hemocytes was only significantly up-regulated at 6 h (8.5-Fold, P < 0.01). RNAi-mediated knockdown of RpSabl transcripts affected the survival rates of Manila clam against V. anguillarum, perhaps mainly due to the inhibited expression of antibacterial effectors (e.g. lysozyme and defensin). Moreover, recombinant protein of RpSabl (rRpSabl) possessed binding activities towards lipopolysaccharides (LPS), peptidoglycan (PGN) and glucan in vitro. Coinciding with the Pathogen-associated molecular patterns (PAMPs) binding assay, rRpSabl displayed broad bacterial-agglutination properties towards Vibrio harveyi, Vibrio splendidus, V. anguillarum, Enterobacter cloacae and Aeromonas hydrophila. Meanwhile, the phagocytosis and encapsulation ability of hemocytes could be significantly enhanced by rRpSabl incubation. All these results showed that RpSabl could function as a versatile molecule involved in the innate immune responses of R. philippinarum.

Detection and characterization of bacterial polysaccharides in drug-resistant enterococci.

Enterococcus faecium (E. faecium) has emerged as one of today's leading causes of health care-associated infections that is difficult to treat with the available antibiotics. These pathogens produce capsular polysaccharides on the cell surface which play a significant role in adhesion, virulence and evasion. Therefore, we aimed at the identification and characterization of bacterial polysaccharide antigens which are central for the development of vaccine-based prophylactic approaches. The crude cell wall-associated polysaccharides from E. faecium, its mutant and complemented strains were purified and analyzed by a primary antibody raised against lipoteichoic acid (LTA) and diheteroglycan (DHG). The resistant E. faecium strains presumably possess novel capsular polysaccharides that allow them to avoid the evasion from opsonic killing. The E. faecium U0317 strain was very well opsonized by anti-U0317 (~95%), an antibody against the whole bacterial cell. The deletion mutant showed a significantly increased susceptibility to opsonophagocytic killing (90-95%) against the penicillin binding protein (anti-PBP-5). By comparison, in a mouse urinary tract and rat endocarditis infection model, respectively, there were no significant differences in virulence. In this study we explored the biological role of the capsule of E. faecium. Our findings showed that the U0317 strain is not only sensitive to anti-LTA but also to antibodies against other enterococcal surface proteins. Our findings demonstrate that polysaccharides capsule mediated-resistance to opsonophagocytosis. We also found that the capsular polysaccharides do not play an important role in bacterial virulence in urinary tract and infective endocarditis in vivo models.

Distinct Spatiotemporal Distribution of Bacterial Toxin-Produced Cellular cAMP Differentially Inhibits Opsonophagocytic Signaling.

Myeloid phagocytes have evolved to rapidly recognize invading pathogens and clear them through opsonophagocytic killing. The adenylate cyclase toxin (CyaA) of Bordetella pertussis and the edema toxin (ET) of Bacillus anthracis are both calmodulin-activated toxins with adenylyl cyclase activity that invade host cells and massively increase the cellular concentrations of a key second messenger molecule, 3',5'-cyclic adenosine monophosphate (cAMP). However, the two toxins differ in the kinetics and mode of cell entry and generate different cAMP concentration gradients within the cell. While CyaA rapidly penetrates cells directly across their plasma membrane, the cellular entry of ET depends on receptor-mediated endocytosis and translocation of the enzymatic subunit across the endosomal membrane. We show that CyaA-generated membrane-proximal cAMP gradient strongly inhibits the activation and phosphorylation of Syk, Vav, and Pyk2, thus inhibiting opsonophagocytosis. By contrast, at similar overall cellular cAMP levels, the ET-generated perinuclear cAMP gradient poorly inhibits the activation and phosphorylation of these signaling proteins. Hence, differences in spatiotemporal distribution of cAMP produced by the two adenylyl cyclase toxins differentially affect the opsonophagocytic signaling in myeloid phagocytes.

Treatment potential of pathogen-reactive antibodies sequentially purified from pooled human immunoglobulin.

OBJECTIVE: Intravenous immune globulin (IVIG), pooled from human blood, is a polyspecific antibody preparation that inhibits the super-antigenic proteins associated with streptococcal and staphylococcal toxic shock, and the Shiga toxin. In addition to this toxin-neutralising activity, IVIG contains other pathogen-reactive antibodies that may confer additional therapeutic benefits. We sought to determine if pathogen-reactive antibodies that promote opsonophagocytosis of different organisms can be sequentially affinity-purified from one IVIG preparation. RESULTS: Antibodies that recognise cell wall antigens of Streptococcus pyogenes, Staphylococcus aureus, and vancomycin-resistant enterococcus (VRE) were sequentially affinity-purified from a single preparation of commercial IVIG and opsonophagocytic activity was assessed using a flow cytometry assay of neutrophil uptake. Non-specific IgG-binding proteins were removed from the S. aureus preparations using an immobilised Fc fragment column, produced using IVIG cleaved with the Immunoglobulin G-degrading enzyme of S. pyogenes (IdeS). Affinity-purified anti-S. aureus and anti-VRE immunoglobulin promoted significantly higher levels of opsonophagocytic uptake by human neutrophils than IVIG when identical total antibody concentrations were compared, confirming activity previously shown for affinity-purified anti-S. pyogenes immunoglobulin. The opsonophagocytic activities of anti-S. pyogenes, anti-S. aureus, and anti-VRE antibodies that were sequentially purified from a single IVIG preparation were undiminished compared to antibodies purified from previously unused IVIG.

Dynamics of formation and morphological features of neutrophil extracellular traps formed under the influence of opsonized Staphylococcus aureus.

In the process of performing their protective functions, neutrophils can form neutrophil extracellular traps (NETs), consisting of DNA in combination with enzymes and histones. The aim of the study was to determine the dynamics of the formation of NETs under the influence of opsonized Staphylococcus aureus and to determine the morphological features of their development in real time by atomic force microscopy. It was found that the maximum formation of NETs was observed after 3 hours of co-incubation of neutrophils and opsonized S. aureus. For the first time, the atomic force microscopy method revealed that, at first, large blocks of parallel DNA helices are formed, which then spread in waves, and only then their bifurcation and separation can be observed. Some of the strands formed are covered by a shell, which subsequently completely disappears. Enzymes and histones become clearly visible only after 140 to 150 minutes of observation. The DNA helixes move toward the opsonized S. aureus. After NET formation, the cell remains on the substrate only in the form of traces of focal adhesion. This, and the fact that the maximum amount of NETs is formed after 3 hours of co-incubation with opsonized S. aureus, suggests that the formation of NETs follows the classical mechanism. The study of the dynamics of formation and the microstructure of NETs makes it possible to estimate the time frame for the implementation of this protective mechanism of the human body when performing the compensatory inflammatory reaction.

Artificial opsonin enhances bacterial phagocytosis, oxidative burst and chemokine production by human neutrophils.

Here, we describe the application of an 'artificial opsonin' to stimulate the innate immune response against Gram-positive bacteria. The artificial opsonin comprises a poly(L-lysine)-graft-poly(ethylene glycol) backbone displaying multiple copies of vancomycin and human IgG-Fc. The vancomycin targets bacteria by recognizing d-Ala-d-Ala-terminated peptides present in the bacterial cell wall. The human IgG-Fc antibody fragments serve as phagocyte recognition moieties that recognize the Fcγ cell surface receptors expressed by professional human phagocytes. Staphylococcus epidermidis RP62A, a biofilm-forming, methicillin-resistant strain, was utilized to investigate the effects of opsonization on phagocytosis, oxidative burst and IL-8 chemokine production by human neutrophils. Results show that opsonization of S. epidermidis RP62A with the artificial opsonin resulted in an ∼2-fold increase in neutrophil phagocytosis. Analysis of the cell supernatant found a 2- to 3-fold increase in neutrophil IL-8 secretion. The neutrophil oxidative burst was investigated using the oxidation-sensitive fluorophore dihydrorhodamine-123. Bacterial opsonization resulted in a 20% increase in fluorescence intensity, indicating a significant increase in the production of reactive oxygen species by the neutrophils. These studies suggest that artificial opsonins may be a novel immunostimulation therapeutic strategy to control infections caused by Gram-positive bacteria, particularly those that are known to be immune evasive and/or antibiotic resistant.

A novel real time imaging platform to quantify macrophage phagocytosis.

Phagocytosis of pathogens, apoptotic cells and debris is a key feature of macrophage function in host defense and tissue homeostasis. Quantification of macrophage phagocytosis in vitro has traditionally been technically challenging. Here we report the optimization and validation of the IncuCyte ZOOM® real time imaging platform for macrophage phagocytosis based on pHrodo® pathogen bioparticles, which only fluoresce when localized in the acidic environment of the phagolysosome. Image analysis and fluorescence quantification were performed with the automated IncuCyte™ Basic Software. Titration of the bioparticle number showed that the system is more sensitive than a spectrofluorometer, as it can detect phagocytosis when using 20× less E. coli bioparticles. We exemplified the power of this real time imaging platform by studying phagocytosis of murine alveolar, bone marrow and peritoneal macrophages. We further demonstrate the ability of this platform to study modulation of the phagocytic process, as pharmacological inhibitors of phagocytosis suppressed bioparticle uptake in a concentration-dependent manner, whereas opsonins augmented phagocytosis. We also investigated the effects of macrophage polarization on E. coli phagocytosis. Bone marrow-derived macrophage (BMDM) priming with M2 stimuli, such as IL-4 and IL-10 resulted in higher engulfment of bioparticles in comparison with M1 polarization. Moreover, we demonstrated that tolerization of BMDMs with lipopolysaccharide (LPS) results in impaired E. coli bioparticle phagocytosis. This novel real time assay will enable researchers to quantify macrophage phagocytosis with a higher degree of accuracy and sensitivity and will allow investigation of limited populations of primary phagocytes in vitro.

Active phagocytosis of Mycobacterium tuberculosis (H37Ra) by T lymphocytes (Jurkat cells).

This study aimed to co-culture Jurkat T lymphocytes with inactivated Mycobacterium tuberculosis (Mtb H37Ra), explore whether T lymphocytes could phagocytose H37Ra cells, and determine the underlying mechanism. Jurkat T lymphocytes were co-cultured with H37Ra cells, and confocal laser scanning microscopy, electron microscopy, and flow cytometry techniques were used to identify phagocytosis and elucidate its mechanism. After Jurkat T lymphocytes phagocytosed H37Ra cells, the cell body became larger, with abundant cytoplasm, the portion of the nucleus closest to the bacterium deformed, long and short pseudopodia were extended, and the folds of the cell membrane formed depressions that created phagocytic vesicles surrounding the bacterium. The macropinocytosis inhibitor amiloride and the cytoskeletal inhibitor cytochalasin D were found to inhibit phagocytic efficacy; serum complements might enhance phagocytosis through opsonization. Jurkat T lymphocytes could actively phagocytose inactivated Mtb via the macropinocytotic mechanism. Actin remodeling played an important role in the macropinocytotic process. Serum complements may regulate phagocytosis.

Quantitative proteome analysis of temporally resolved phagosomes following uptake via key phagocytic receptors.

Macrophages operate at the forefront of innate immunity and their discrimination of foreign versus "self" particles is critical for a number of responses including efficient pathogen killing, antigen presentation, and cytokine induction. In order to efficiently destroy the particles and detect potential threats, macrophages express an array of receptors to sense and phagocytose prey particles. In this study, we accurately quantified a proteomic time-course of isolated phagosomes from murine bone marrow-derived macrophages induced by particles conjugated to seven different ligands representing pathogen-associated molecular patterns, immune opsonins or apoptotic cell markers. We identified a clear functional differentiation over the three timepoints and detected subtle differences between certain ligand-phagosomes, indicating that triggering of receptors through a single ligand type has mild, but distinct, effects on phagosome proteome and function. Moreover, our data shows that uptake of phosphatidylserine-coated beads induces an active repression of NF-κB immune responses upon Toll-like receptor (TLR)-activation by recruitment of anti-inflammatory regulators to the phagosome. This data shows for the first time a systematic time-course analysis of bone marrow-derived macrophages phagosomes and how phagosome fate is regulated by the receptors triggered for phagocytosis.

Antioxidant, antimicrobial and neutrophil-modulating activities of herb extracts.

The present study provides a comprehensive data on the antioxidant, antimicrobial and neutrophil-modulating activities of extracts from six medicinal plants--blackberry (Rubus fruticosus) leaves, chokeberry (Aronia melanocarpa) leaves, hawthorn (Crataegus monogyna) leaves, lady's mantle (Alchemilla glabra) aerial parts, meadowsweet (Filipendula ulmaria) aerial parts and raspberry (Rubus idaeus) leaves. In order to analyze the antioxidant activity of the herbs, several methods (ORAC, TRAP, HORAC and inhibition of lipid peroxidation) were used. Blackberry leaves and meadowsweet extracts revealed the highest antioxidant activities via all methods. All extracts studied blocked almost completely the opsonized zymosan particle-activated ROS production by neutrophils from human whole blood. On the other hand, the effect of extracts on phorbol myristate acetate-activated ROS production was much milder and even nonsignificant in the case of chokeberry leaves. This latter result suggests that extracts (apart from their antioxidative activity) interfere with the signaling cascade of phagocyte activation upstream of the protein kinase C activation. The antimicrobial activity of the investigated extracts against 11 human pathogens was investigated using three different methods. Meadowsweet and blackberry leaves extracts had the highest antimicrobial effect and the lowest minimal inhibiting concentrations (MICs) against the microorganisms tested.

Adiponectin inhibits neutrophil phagocytosis of Escherichia coli by inhibition of PKB and ERK 1/2 MAPK signalling and Mac-1 activation.

Full length adiponectin is a potent immune modulatory adipokine, impacting upon the actions of several immune cells. Neutrophil oxidative burst has been shown to decrease in response to adiponectin, and we speculated that it could have other effects on neutrophil function. Here we report that adiponectin reduces the phagocytic ability of human neutrophils, decreasing significantly the ingestion of opsonised E. coli by these cells in whole blood (p<0.05) and as isolated neutrophils (p<0.05). We then determined the mechanisms involved. We observed that the activation of Mac-1, the receptor engaged in complement-mediated phagocytosis, was decreased by adiponectin in response to E. coli stimulation. Moreover, treatment of neutrophils with adiponectin prior to incubation with E. coli significantly inhibited signalling through the PI3K/PKB and ERK 1/2 pathways, with a parallel reduction of F-actin content. Studies with pharmacological inhibitors showed that inhibition of PI3K/PKB, but not ERK 1/2 signalling was able to prevent the activation of Mac-1. In conclusion, we propose that adiponectin negatively affects neutrophil phagocytosis, reducing the uptake of E. coli and inhibiting Mac-1 activation, the latter by blockade of the PI3K/PKB signal pathway.

Antibodies Against Sporothrix schenckii Enhance TNF-α Production and Killing by Macrophages.

Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungus Sporothrix schenckii. The immunological mechanisms involved in the prevention and control of sporotrichosis suggest that cell-mediated immunity plays an important role in protecting the host against S. schenckii. Nonetheless, recent data strongly support the existence of protective Abs against this pathogenic fungus. In a previous study, we showed that passive Ab therapy led to a significant reduction in the number of colony forming unit in the organs of mice when the MAb was injected before and during S. schenckii infection. The ability of opsonization to enhance macrophage damage to S. schenckii and subsequent cytokine production was investigated in this work. Here we show that the fungicidal characteristics of macrophages are increased when the fungus is phagocytosed in the presence of inactivated serum from mice infected with S. schenckii or mAb anti-gp70. Additionally, we show an increase in the levels of pro-inflammatory cytokines such as TNF-α and IL-1ß. This study provides additional support for the importance of antibodies in protecting against S. schenckii and concludes that opsonization is an important process to increase TNF-α production and fungus killing by macrophages in experimental sporotrichosis.

Antigenic modulation and rituximab resistance.

Several types of B-cell lymphoma have been successfully treated with rituximab, and approval by the US Food and Drug Administration for use of rituximab in the treatment of rheumatoid arthritis has increased interest in targeting CD20 on B cells for other indications. Although large amounts of rituximab can be infused into humans with no apparent dose-limiting toxicity, recent evidence suggests that the body's effector mechanisms, including complement-mediated cytotoxicity and natural killer (NK) cell-mediated killing, can be saturated or exhausted at high burdens of rituximab-opsonized B cells. One of the consequences of this saturation phenomenon is that the opsonized B cells are instead processed by a different pathway mediated by FcgammaR on effector cells. In this alternative pathway, both rituximab and CD20 are removed ("shaved") from the B cells and are taken up by monocytes/macrophages. This process, formerly called antigenic modulation, appears to occur in several compartments in the body and may play a key role in the development of resistance to rituximab therapy.

Dysopsonin activity of serum DNA-binding proteins favorable for gene delivery.

Naked DNA is regarded as the safest and simplest method of gene delivery. However, normally intravenously injected naked plasmid DNA is rapidly eliminated from the blood. It has been hypothesized that opsonins, a category of serum DNA-binding proteins (SDBPs), label the injected plasmid DNA as foreign so that it may be recognized and rapidly removed from the bloodstream by liver nonparenchymal cells. Contrary to the hypothesis, our data indicate that some SDBPs across multiple species may have important dysopsonin properties, acting to reduce liver uptake. Formation of SDBP and DNA complexes was observed by agarose gel electrophoresis. An in vivo study involving hepatic artery and portal vein occlusion in a mouse model confirmed the activity of serum diminishing liver uptake of DNA. Data using hydrodynamic gene transfer in the mouse liver and in situ transfection in the mouse lung revealed that serum proteins bound to DNA do not affect the biological activity of the plasmid DNA. We have identified several SDBPs with potential dysopsonin properties. The SDBPs with dysopsonin properties and DNA complexes may be further modified and ultimately be developed into a novel DNA carrier system favorable for systemic gene delivery.

M2 macrophages phagocytose rituximab-opsonized leukemic targets more efficiently than m1 cells in vitro.

Because macrophages have been implicated as major players in the mechanism of action of rituximab, we have investigated the factors that modulate their tumor cell killing potential. Human macrophages, differentiated in vitro from peripheral blood monocytes, were used in binding and phagocytosis assays using B-chronic lymphocytic leukemia or lymphoma target cells opsonized with rituximab. Phagocytosis was maximal at 0.1 microg/ml rituximab and was not significantly affected by CD20 expression levels or by CD16A polymorphism at position 158 (Val/Phe). The role of FcgammaRs was demonstrated by complete inhibition of phagocytosis by excess human Igs. Because macrophages can be differentiated to M1- or M2-type cells with either GM-CSF or M-CSF, respectively, and can be classically activated by proinflammatory stimuli (IFN-gamma/LPS) or undergo alternative activation with cytokines such as IL-4 or IL-10, we have analyzed the effect of these different polarization programs on the phagocytosis mediated by rituximab. Macrophages differentiated in presence of M-CSF showed a 2- to 3-fold greater phagocytic capacity compared with GM-CSF-induced cells. Furthermore, addition of IL-10 significantly increased, whereas IL-4 decreased phagocytosis by both M-CSF- and GM-CSF-differentiated macrophages. LPS/IFN-gamma had little effect. Expression of CD16, CD32, and CD64 in different macrophage populations correlated with phagocytic activity. Interestingly, several B lymphoma cell lines were observed to secrete 400-1300 pg/ml IL-10 in vitro, and coculture of human macrophages with lymphoma conditioned medium increased significantly their phagocytic capacity. Our data suggest that cytokines secreted by lymphoma cells can favor alternate activation of macrophages with a high phagocytic capacity toward rituximab-opsonized targets.

Manno(rhamno)biose-containing capsular polysaccharides of Klebsiella pneumoniae enhance opsono-stimulation of human polymorphonuclear leukocytes.

We tested the relationship between the capsular and the O-antigen structures and the ability of bacteria to trigger respiratory burst in human polymorphonuclear leukocytes (PMNL). Capsulated and non-capsulated variants as well as capsule-switched derivatives of Klebsiella serotypes bearing or lacking manno(rhamno)biose repeats in their capsular polysaccharides and expressing either mannose-rich or mannose-poor O antigens were tested for their ability to induce respiratory burst and survive in human PMNL. Luminol-enhanced chemiluminescence (CL) was measured to quantify respiratory burst. Intracellular survival was quantified by determining the viable counts of intracellular bacteria. K serotypes and the capsule-switched derivative lacking manno(rhamno)biose induced significantly lower CL than those expressing manno(rhamno)biose. Manno(rhamno)biose-lacking serotypes survived in the cells significantly better than serotypes expressing these repeats. C1q depletion did not affect CL induced by the manno(rhamno)biose-containing serotype, whereas factor B depletion revealed a significantly reduced CL. Likewise, EGTA in the presence of Mg(2+) significantly decreased CL, but the values were higher than those induced by the bacterium opsonized with factor B-depleted serum. In the presence of EGTA, Mg(2+)-treated factor B-depleted serum revealed a significant reduction in the CL response compared with the responses induced by opsonization with factor B-depleted serum alone. These results indicate, in addition to the alternative pathway, a manno(rhamno)biose pattern recognition of Klebsiella by PMNL probably by the complement lectin pathway.