Effects of iloperidone on hERG 1A/3.1 heterotetrameric channels.

OBJECTIVES: Iloperidone is an atypical antipsychotic drug that is widely used for the treatment of schizophrenia. hERG 3.1, alternatively spliced form of hERG 1A, is considered a potential target for an antipsychotic drug. The present study was designed to study the effects of iloperidone on hERG 1A/3.1 heterotetrameric channels. METHODS: The interactions of iloperidone with hERG 1A/3.1 heterotetrameric channels stably expressed in HEK cells were investigated using the whole-cell patch-clamp technique and western blot analysis. RESULTS: Iloperidone inhibited the hERG 1A/3.1 tail currents at -50 mV in a concentration-dependent manner with an IC50 value of 0.44 µM. The block of hERG 1A/3.1 currents by iloperidone was voltage-dependent and increased over a range of voltage for channel activation. However, the block by iloperidone was voltage-independent at more depolarized potentials where the channels were fully activated. A fast application of iloperidone inhibited the hERG 1A/3.1 current elicited by a 5-s depolarizing pulse to +60 mV to fully inactivate the hERG 1A/3.1 currents. Iloperidone also induced a rapid and reversible inhibition of hERG 1A/3.1 tail currents during repolarization. However, iloperidone had no effect on either hERG 1A or hERG 1A/3.1 channel trafficking to the cell membrane. CONCLUSIONS: Our results indicated that iloperidone concentration-dependently inhibited hERG 1A/3.1 currents by preferentially interacting with the open states of channels, but not by the disruption of membrane trafficking or surface membrane expression of hERG 1A and hERG 1A/3.1 channel proteins.

APEX Proximity Labeling as a Versatile Tool for Biological Research.

Most proteins are specifically localized in membrane-encapsulated organelles or non-membrane-bound compartments. The subcellular localization of proteins facilitates their functions and integration into functional networks; therefore, protein localization is tightly regulated in diverse biological contexts. However, protein localization has been mainly analyzed through immunohistochemistry or the fractionation of subcellular compartments, each of which has major drawbacks. Immunohistochemistry can examine only a handful of proteins at a time, and fractionation inevitably relies on the lysis of cells, which disrupts native cellular conditions. Recently, an engineered ascorbate peroxidase (APEX)-based proximity labeling technique combined with mass spectrometry was developed, which allows for temporally and spatially resolved proteomic mapping. In the presence of H2O2, engineered APEX oxidizes biotin-phenols into biotin-phenoxyl radicals, and these short-lived radicals biotinylate electron-rich amino acids within a radius of several nanometers. Biotinylated proteins are subsequently enriched by streptavidin and identified by mass spectrometry. This permits the sensitive and efficient labeling of proximal proteins around locally expressed APEX. Through the targeted expression of APEX in the subcellular region of interest, proteomic profiling of submitochondrial spaces, the outer mitochondrial membrane, the endoplasmic reticulum (ER)-mitochondrial contact, and the ER membrane has been performed. Furthermore, this method has been modified to define interaction networks in the vicinity of target proteins and has also been applied to analyze the spatial transcriptome. In this Perspective, we provide an outline of this newly developed technique and discuss its potential applications to address diverse biological questions.

Targeting protein biotinylation enhances tuberculosis chemotherapy.

Successful drug treatment for tuberculosis (TB) depends on the unique contributions of its component drugs. Drug resistance poses a threat to the efficacy of individual drugs and the regimens to which they contribute. Biologically and chemically validated targets capable of replacing individual components of current TB chemotherapy are a major unmet need in TB drug development. We demonstrate that chemical inhibition of the bacterial biotin protein ligase (BPL) with the inhibitor Bio-AMS (5'-[N-(d-biotinoyl)sulfamoyl]amino-5'-deoxyadenosine) killed Mycobacterium tuberculosis (Mtb), the bacterial pathogen causing TB. We also show that genetic silencing of BPL eliminated the pathogen efficiently from mice during acute and chronic infection with Mtb Partial chemical inactivation of BPL increased the potency of two first-line drugs, rifampicin and ethambutol, and genetic interference with protein biotinylation accelerated clearance of Mtb from mouse lungs and spleens by rifampicin. These studies validate BPL as a potential drug target that could serve as an alternate frontline target in the development of new drugs against Mtb.

Proteomic analysis of the cullin 4B interactome using proximity-dependent biotinylation in living cells.

Cullin 4B (CUL4B) mutations have been implicated in mental retardation and dopamine-related behaviors due to disruptions in their interaction with cullin-RING E3 ligases (CRLs). Thus, further identification of CUL4B substrates can increase the knowledge of protein homeostasis and illuminate the role of CUL4B in neuropsychiatric disease. However, the transient nature of the coupling between CUL4B and its substrates is difficult to detect in vivo using current approaches, thus hampers efforts to investigate functions of CRLs within unperturbed living systems. In this study, we sought to discover CUL4B interactants with or without dopamine stimulation. BirA (118G) proximity-dependent biotin labeling combined with LC-MS was employed to biotinylate and identify transient and weak interactants of CUL4B. After purification with streptavidin beads and identified by LC-MS, a total of 150 biotinylated proteins were identified at baseline condition, 53 of which are well-known CUL4B interactants. After dopamine stimulation, 29 proteins disappeared and were replaced by 21 different protein interactants. The altered CUL4B interactants suggest that CUL4B regulates protein turnover and homeostasis in response to dopamine stimulation. Our results demonstrate the potential of this approach to identify novel CUL4B-related molecules in respond to cellular stimuli, which may be applied to other types of signaling pathways.

A novel regulatory role for tissue transglutaminase in epithelial-mesenchymal transition in cystic fibrosis.

Cystic fibrosis (CF) is a genetic disorder caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) for which there is no overall effective treatment. Recent work indicates tissue transglutaminase (TG2) plays a pivotal intracellular role in proteostasis in CF epithelia and that the pan TG inhibitor cysteamine improves CFTR stability. Here we show TG2 has another role in CF pathology linked with TGFß1 activation and signalling, induction of epithelial-mesenchymal transition (EMT), CFTR stability and induction of matrix deposition. We show that increased TG2 expression in normal and CF bronchial epithelial cells increases TGFß1 levels, promoting EMT progression, and impairs tight junctions as measured by Transepithelial Electric Resistance (TEER) which can be reversed by selective inhibition of TG2 with an observed increase in CFTR stability. Our data indicate that selective inhibition of TG2 provides a potential therapeutic avenue for reducing fibrosis and increasing CFTR stability in CF.

Ultrasound-mediated gene transfer (sonoporation) in fibrin-based matrices: potential for use in tissue regeneration.

It has been suggested that gene transfer into donor cells is an efficient and practical means of locally supplying requisite growth factors for applications in tissue regeneration. Here we describe, for the first time, an ultrasound-mediated system that can non-invasively facilitate gene transfer into cells entrapped within fibrin-based matrices. Since ultrasound-mediated gene transfer is enhanced using microbubbles, we compared the efficacy of neutral and cationic forms of these reagents on the ultrasound-stimulated gene transfer process in gel matrices. In doing so we demonstrated the beneficial effects associated with the use of cationic microbubble preparations that interact directly with cells and nucleic acid within matrices. In some cases, gene expression was increased two-fold in gel matrices when cationic microbubbles were compared with neutral microbubbles. In addition, incorporating collagen into fibrin gels yielded a 25-fold increase in gene expression after application of ultrasound to microbubble-containing matrices. We suggest that this novel system may facilitate non-invasive temporal and spatial control of gene transfer in gel-based matrices for the purposes of tissue regeneration.

Resveratrol compounds inhibit human holocarboxylase synthetase and cause a lean phenotype in Drosophila melanogaster.

Holocarboxylase synthetase (HLCS) is the sole protein-biotin ligase in the human proteome. HLCS has key regulatory functions in intermediary metabolism, including fatty acid metabolism, and in gene repression through epigenetic mechanisms. The objective of this study was to identify food-borne inhibitors of HLCS that alter HLCS-dependent pathways in metabolism and gene regulation. When libraries of extracts from natural products and chemically pure compounds were screened for HLCS inhibitor activity, resveratrol compounds in grape materials caused an HLCS inhibition of >98% in vitro. The potency of these compounds was piceatannol>resveratrol>piceid. Grape-borne compounds other than resveratrol metabolites also contributed toward HLCS inhibition, e.g., p-coumaric acid and cyanidin chloride. HLCS inhibitors had meaningful effects on body fat mass. When Drosophila melanogaster brummer mutants, which are genetically predisposed to storing excess amounts of lipids, were fed diets enriched with grape leaf extracts and piceid, body fat mass decreased by more than 30% in males and females. However, Drosophila responded to inhibitor treatment with an increase in the expression of HLCS, which elicited an increase in the abundance of biotinylated carboxylases in vivo. We conclude that mechanisms other than inhibition of HLCS cause body fat loss in flies. We propose that the primary candidate is the inhibition of the insulin receptor/Akt signaling pathway.

Regulation of immunological and inflammatory functions by biotin.

Biotin is a water-soluble B-complex vitamin and is well-known as a co-factor for 5 indispensable carboxylases. Holocarboxylase synthetase (HLCS) catalyzes the biotinylation of carboxylases and other proteins, whereas biotinidase catalyzes the release of biotin from biotinylated peptides. Previous studies have reported that nutritional biotin deficiency and genetic defects in either HLCS or biotinidase induces cutaneous inflammation and immunological disorders. Since biotin-dependent carboxylases involve various cellular metabolic pathways including gluconeogenesis, fatty acid synthesis, and the metabolism of branched-chain amino acids and odd-chain fatty acids, metabolic abnormalities may play important roles in immunological and inflammatory disorders caused by biotin deficiency. Transcriptional factors, including NF-κB and Sp1/3, are also affected by the status of biotin, indicating that biotin regulates immunological and inflammatory functions independently of biotin-dependent carboxylases. An in-vivo analysis with a murine model revealed the therapeutic effects of biotin supplementation on metal allergies. The novel roles of biotinylated proteins and their related enzymes have recently been reported. Non-carboxylase biotinylated proteins induce chemokine production. HLCS is a nuclear protein involved in epigenetic and chromatin regulation. In this review, comprehensive knowledge on the regulation of immunological and inflammatory functions by biotin and its potential as a therapeutic agent is discussed.

Quantifying the bioadhesive properties of surface-modified polyurethane-urea nanoparticles in the vascular network.

Nanomedicine research is currently requiring new standard methods to quantify the biocompatibility and bioadhesivity of emerging biomaterials designed to be used in contact with blood or soft tissues. In this study, we used biotinylated polyurethane-urea nanoparticles as a model to examine the applicabitility of an adapted hemagglutination assay to quantify the bioadhesive potential of these nanoparticles to red blood cells and, in turn, to extrapolate this data to vascular endothelial cells. We demonstrated that biotinylated nanoparticles adsorb to human erythrocytes and preferentially gather in erythrocyte contact areas. Moreover, these nanoparticles promoted a higher percentage of pig and human erythrocyte agglutination than naked polyurethane-urea nanoparticles in a biotin concentration-dependent manner. Conversely, pegylated nanoparticles were used as a negative control of the technique thus showing decreasing hemagglutination values as compared to naked nanoparticles until a minimum threshold. Furthermore, hemagglutination assay demonstrated an excellent positive correlation with bioadhesion quantification in human endothelial cells and the endothelial layer of pig aorta thus validating the hemagglutination assay described here as a useful method for predicting nanoparticle bioadhesivity to vascular endothelium. Therefore, the methodology described here is a versatile and straightforward method that allows evaluating the bioadhesive features of surface-modified polyurethane-urea nanoparticles in contact with blood and the vascular network and appears as a powerful tool to better design any drug delivery systems or implantable devices for biomedical applications.

Synthesis and biological evaluation of dual action cyclo-RGD/SMAC mimetic conjugates targeting α(v)ß(3)/α(v)ß(5) integrins and IAP proteins.

The rational design, synthesis and in vitro biological evaluation of dual action conjugates 11-13, containing a tumour targeting, integrin αvß3/αvß5 ligand portion and a pro-apoptotic SMAC mimetic portion (cyclo-RGD/SMAC mimetic conjugates) are reported. The binding strength of the two separate units is generally maintained by these dual action conjugates. In particular, the connection between the separate units (anchor points on each unit; nature, length and stability of the linker) influences the activity of each portion against its molecular targets (integrins αvß3/αvß5 for cyclo-RGD, IAP proteins for SMAC mimetics). Each conjugate portion tolerates different substitutions while preserving the binding affinity for each target.

Investigating dengue virus nonstructural protein 5 (NS5) nuclear import.

Dengue virus (DENV) nonstructural protein 5 (NS5) plays a central role in viral replication in the cytoplasm of infected cells. Despite this, NS5 is predominantly located in the nucleus of infected cells where it is thought to play a role in suppression of the host antiviral response. We have investigated the nuclear localization of NS5 using immunofluorescent staining for NS5 in infected cells, showing that NS5 nuclear localization is significantly inhibited by Ivermectin, a general inhibitor of nuclear transport mediated by the cellular nuclear transport proteins importin α/ß (IMPα/ß). Experiments in living mammalian cells transfected to express green fluorescent protein (GFP)-tagged NS5 protein confirm that NS5 is predominantly nuclear and that this localization is inhibited by Ivermectin, demonstrating that NS5 contains an Ivermectin-sensitive IMPα/ß-recognized nuclear localization signal [Pryor et al. Traffic 8:795-807, 2007]. Consistent with this observation, mutation of critical residues within the nuclear localization signal (the A2 mutant; [Pryor et al. Traffic 8:795-807, 2007]) results in an 80 % reduction in nuclear localization of NS5. Finally we demonstrate direct, high-affinity binding of NS5 to IMPα/ß using an AlphaScreen protein-protein binding assay.

Rab14 regulation of claudin-2 trafficking modulates epithelial permeability and lumen morphogenesis.

Regulation of epithelial barrier function requires targeted insertion of tight junction proteins that have distinct selectively permeable characteristics. The insertion of newly synthesized proteins and recycling of internalized tight junction components control both polarity and junction function. Here we show that the small GTPase Rab14 regulates tight junction structure. In Madin-Darby canine kidney (MDCK) II cells, Rab14 colocalizes with junctional proteins, and knockdown of Rab14 results in increased transepithelial resistance. In cells without Rab14, there are small changes in the trafficking of claudin-1 and occludin. In addition, there is substantial depletion of the leaky claudin, claudin-2, but not other tight junction components. The loss of claudin-2 is complemented by inhibition of lysosomal function, suggesting that Rab14 sorts claudin-2 out of the lysosome-directed pathway. MDCK I cells lack claudin-2 endogenously, and knockdown of Rab14 in these cells does not result in a change in transepithelial resistance, suggesting that the effect is specific to claudin-2 trafficking. Furthermore, leaky claudins have been shown to be required for epithelial morphogenesis, and knockdown of Rab14 results in failure to form normal single-lumen cysts in three-dimensional culture. These results implicate Rab14 in specialized trafficking of claudin-2 from the recycling endosome.

Reconstituting functional microtubule-barrier interactions.

Local interactions between the tips of microtubules and the cell cortex, or other cellular components such as kinetochores, play an important role in essential cellular processes like establishing cell polarity, distribution of organelles, and microtubule aster and chromosome positioning. Here we present two in vitro assays that specifically mimic microtubule-cortex interactions by employing selectively functionalized microfabricated barriers that allow for the immobilization of proteins with a range of affinities. We describe the microfabrication process to create gold or glass barriers and the subsequent functionalization of these barriers using self-assembled thiol monolayers or polylysine-poly(ethylene glycol), respectively. Near-permanent attachment of proteins is obtained using biotinylated surfaces combined with streptavidin and biotinylated proteins. Lower affinity interactions, further tunable with the addition of imidazole, are obtained using nickel-nitrilotriacetic acid (Ni(II)-NTA) functionalization combined with his-tagged proteins. Both mono-NTA and tris-NTA compounds are used. We show an assay to reconstitute the "end-on" interaction between dynamic microtubule tips and barrier-attached dynein, mimicking the cellular situation at the cortex and at kinetochores. In a second assay, we reconstitute microtubule-based delivery of end-tracking proteins to functionalized barriers, mimicking the transport of cell-end markers to the cell poles in interphase fission yeast cells.

Characterization of a monoclonal antibody capable of reliably quantifying expression of human Copper Transporter 1 (hCTR1).

Human copper transporter 1 (hCTR1) is the high-affinity copper influx transporter in mammalian cells that also mediates the influx of cisplatin. Loss of hCTR1 expression has been implicated in the development of resistance to this cancer chemotherapeutic agent. It has turned out to be very difficult to develop antibodies to hCTR1 and polyclonal antibodies produced by different laboratories have yielded conflicting results. We have characterized a newly-available rabbit monoclonal antibody that reacts with an epitope on the N-terminal end of hCTR1 that now permits rigorous identification and quantification of hCTR1 using Western blot analysis. Postnuclear membrane (PNM) preparations made from cells engineered to express high levels of myc-tagged hCTR1, and cells in which the expression of hCTR1 was knocked down, were used to characterize the antibody. The identity of the bands detected was confirmed by immunoprecipitation, surface biotinylation and deglycosylation of myc-tagged hCTR1. Despite the specificity expected of a monoclonal antibody, the anti-hCTR1 detected a variety of bands in whole cell lysates (WCL), which made it difficult to quantify hCTR1. This problem was overcome by isolating post-nuclear membranes and using these for further analysis. Three bands were identified using this antibody in PNM preparations that migrated at 28, 33-35 and 62-64kDa. Multiple lines of evidence presented here suggest that the 33-35 and 62-64kDa bands are hCTR1 whereas the 28kDa band is a cross-reacting protein of unknown identify. The 33-35kDa band is consistent with the expected MW of the glycosylated hCTR1 monomer. This analysis now permits rigorous identification and quantification of hCTR1.

Design, fabrication and in vitro evaluation of a novel polymer-hydrogel hybrid scaffold for bone tissue engineering.

The development of a bone mechanically-compatible and osteoinductive scaffold is important for bone tissue engineering applications, particularly for the repair and regeneration of large area critically-sized bone defects. Although previous studies with weight-bearing scaffolds have shown promising results, there is a clear need to develop better osteoinductive strategies for effective scaffold-based bone regeneration. In this study, we designed and fabricated a novel polymer-hydrogel hybrid scaffold system in which a load-bearing polymer matrix and a peptide hydrogel allowed for the synergistic combination of mechanical strength and great potential for osteoinductivity in a single scaffold. The hybrid scaffold system promoted increased pre-osteoblastic cell proliferation. Further, we biotinylated human recombinant bone morphogenetic protein 2 (rhBMP2), and characterized the biotin addition and its effect on rhBMP2 biological activity. The biotinylated rhBMP2 was tethered to the hybrid scaffold using biotin-streptavidin complexation. Controlled release studies demonstrated increased rhBMP2 retention with the tethered rhBMP2 hybrid scaffold group. In vitro evaluation of the hybrid scaffold was performed with rat bone marrow stromal cells and mouse pre-osteoblast cell line MC3T3-E1 cells. Gene expression of alkaline phosphatase (ALP), collagen I (Col I), osteopontin (OPN), bone sialoprotein (BSP), Runx-2 and osteocalcin (OC) increased in MC3T3-E1 cells seeded on the rhBMP2 tethered hybrid scaffolds over the untethered counterparts, demonstrating osteoinductive potential of the hybrid graft. These findings suggest the possibility of developing a novel polymer-hydrogel hybrid system that is weight bearing and osteoinductive for effective bone tissue engineering.

The streptavidin/biotinylated DNA/protein bound complex protocol for determining the association of c-JUN protein with NANOG promoter.

Chromatin immunoprecipitation (ChIP) is a widely used and pre-eminent technique for detecting the association of an individual protein or a particular protein complex with its specific DNA sequence(s) in vivo. Herein we introduce a novel and simple biotinylated-oligonucleotide-mediated ChIP method for testing specific binding of the c-JUN protein to the M1-DNA-regulatory element in the NANOG promoter. We prepared a 260-bp DNA PCR amplicon containing -300 bp to -59 bp, relative to the transcriptional start site of the human NANOG gene, which was transfected into mouse embryonic fibroblasts (MEF) containing wild-type (c-jun(+/+)) or knockout c-jun (c-jun(-/-)) alleles. Whole cells that were cross-linked using formaldehyde and protein-DNA interactions were immunoprecipitated using streptavidin-coupled Dynabeads. Protein-DNA cross-links were reversed during incubation at 95°C, and protein samples were visualized using SDS-PAGE electrophoresis and western blotting. This streptavidin/biotinylated DNA/protein-bound complex protocol can be used for detecting the interactions between multiple transcription factors and their DNA binding sites.

Regulatable in vivo biotinylation expression system in mouse embryonic stem cells.

Embryonic stem (ES) cells have several unique attributes, the two most important of which are they can differentiate into all cell types in the body and they can proliferate indefinitely. To study the regulation of these phenomena, we developed a regulatable in vivo biotinylation expression system in mouse ES cells. The E. coli biotin ligase gene BirA, whose protein product can biotinylate a 15-aa peptide sequence, called the AviTag, was cloned downstream of an IRES. The primary vector containing the doxycycline controlled transactivator gene tTA and IRES-BirA was knocked into the ROSA26 locus by homologous recombination. The secondary vector containing the AviTag tagged hKlf4 gene was exchanged into the ROSA26 locus using Cre recombinase. Western blot analysis showed that the doxycycline induced BirA protein can biotinylate the doxycycline induced AviTag tagged hKlf4 protein. The induction of hKlf4 repressed cell growth in the presence or absence of LIF. Chromatin immunoprecipitation assays using streptavidin beads showed that the AviTag tagged hKlf4 protein could enrich the Nanog enhancer. Our results suggested that the regulatable biotinylation system is promising for the gene function studies in mouse ES cells.

Amyloid fibrils composed of hexameric peptides attenuate neuroinflammation.

The amyloid-forming proteins tau, αB crystallin, and amyloid P protein are all found in lesions of multiple sclerosis (MS). Our previous work established that amyloidogenic peptides from the small heat shock protein αB crystallin (HspB5) and from amyloid ß fibrils, characteristic of Alzheimer's disease, were therapeutic in experimental autoimmune encephalomyelitis (EAE), reflecting aspects of the pathology of MS. To understand the molecular basis for the therapeutic effect, we showed a set of amyloidogenic peptides composed of six amino acids, including those from tau, amyloid ß A4, major prion protein (PrP), HspB5, amylin, serum amyloid P, and insulin B chain, to be anti-inflammatory and capable of reducing serological levels of interleukin-6 and attenuating paralysis in EAE. The chaperone function of the fibrils correlates with the therapeutic outcome. Fibrils composed of tau 623-628 precipitated 49 plasma proteins, including apolipoprotein B-100, clusterin, transthyretin, and complement C3, supporting the hypothesis that the fibrils are active biological agents. Amyloid fibrils thus may provide benefit in MS and other neuroinflammatory disorders.

Bioengineered surfaces to improve the blood compatibility of biomaterials through direct thrombin inactivation.

Thrombus formation, due to thrombin generation, is a major problem affecting blood-contacting medical devices. This work aimed to develop a new strategy to improve the hemocompatibility of such devices by the immobilization of a naturally occurring thrombin inhibitor into a nanostructured surface. Boophilin, a direct thrombin inhibitor from the cattle tick Rhipicephalus microplus, was produced as a recombinant protein in Pichia pastoris. Boophilin was biotinylated and immobilized on biotin-terminated self-assembled monolayers (SAM) via neutravidin. In order to maintain its proteinase inhibitory capacity after surface immobilization, boophilin was biotinylated after the formation of a boophilin-thrombin complex to minimize the biotinylation of the residues involved in thrombin-boophilin interaction. The extent of boophilin biotinylation was determined using matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry. Boophilin immobilization and thrombin adsorption were quantified using quartz crystal microbalance with dissipation. Thrombin competitive adsorption from human serum was assessed using ¹²5I-thrombin. Thrombin inhibition and plasma clotting time were determined using spectrophotometric techniques. Boophilin-coated SAM were able to promote thrombin adsorption in a selective way, inhibiting most of its activity and delaying plasma coagulation in comparison with boophilin-free surfaces, demonstrating boophilin's potential to improve the hemocompatibility of biomaterials used in the production of blood-contacting devices.

The inhibition of anti-DNA binding to DNA by nucleic acid binding polymers.

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE) and can mediate disease pathogenesis by the formation of immune complexes. Since blocking immune complex formation can attenuate disease manifestations, the effects of nucleic acid binding polymers (NABPs) on anti-DNA binding in vitro were investigated. The compounds tested included polyamidoamine dendrimer, 1,4-diaminobutane core, generation 3.0 (PAMAM-G3), hexadimethrine bromide, and a ß-cylodextrin-containing polycation. As shown with plasma from patients with SLE, NABPs can inhibit anti-DNA antibody binding in ELISA assays. The inhibition was specific since the NABPs did not affect binding to tetanus toxoid or the Sm protein, another lupus autoantigen. Furthermore, the polymers could displace antibody from preformed complexes. Together, these results indicate that NABPs can inhibit the formation of immune complexes and may represent a new approach to treatment.