The immune complex transfer enzyme immunoassay: Mechanism of improved sensitivity compared with conventional sandwich enzyme immunoassay.

Immune complex transfer enzyme immunoassay (ICT-EIA) is one of the technologies which enables ultrasensitive measurements of protein biomarkers. The ICT-EIA uses two types of beads and sandwich-shaped immune complexes are transferred from the 1st bead to the 2nd bead in the assay. The purpose of the study is to reveal the reason why the ICT-EIA achieves ultrasensitive measurements by making a detailed comparison between conventional sandwich enzyme immunoassay (Sand-EIA) and ICT-EIA. ICT-EIAs for cytokines were developed and the sensitivities were compared with the sandwich EIAs. ICT-EIAs had about 100 times higher sensitivities because of markedly decreased non-specific signals derived from non-specific binding of detection antibody conjugates onto the polystyrene bead. The results have enabled us to show the importance of reducing non-specific signals in EIAs to obtain higher sensitivities. This methodology should be more valuable if combined with a different label detection system such as digital counting or immuno-PCR, which may enable the detection of single target protein molecules in the near future.

Mechanism of nanoparticle-induced hypersensitivity in pigs: complement or not complement?

A recent study on nanoparticle-induced hypersensitivity reactions in pigs showed robust pulmonary intravascular macrophage clearance of Polybead® carboxylate microspheres in mediating the adverse cardiopulmonary distress, irrespective of the ability of these particles to activate the complement (C) system in vitro. Focusing on this observation, this article highlights the controversies in projecting in vitro C assay data to in vivo conditions and applying data on polystyrene particles to therapeutic nanopharmaceuticals. Based on overwhelming evidence of a role of anaphylatoxins in hypersensitivity reactions, the need to further explore the role of C activation in the reported and other reactions is highlighted. C-activation-related and C-independent pseudoallergies (CARPA and CIPA) can proceed simultaneously, as outlined by the 'double-hit' hypothesis.

Immunogenicity of antigen-conjugated biodegradable polydiacetylene liposomes administered mucosally.

In this work, we report a protocol for synthesizing nanosize ovalbumin-functionalized polydiacetylene (PDA) liposomes (LP-Ova). We show that LP-Ova administered per-orally (p.o.) and subcutaneously (s.c.), without the use of adjuvants, induces high serum IgG1 titers. As reported previously using polystyrene nanoparticles (NPs), p.o.-primed mice developed high titers of IgG2c and intestinal IgA following s.c. boosting immunization with LP-Ova. Mice that received a single s.c. immunization with LP-Ova did not develop serum IgG2c or intestinal IgA antibodies. Additionally, in s.c.-immunized mice serum IgG1 titers decreased significantly by 3 months after immunization. In contrast, in mice primed p.o. and boosted s.c. with LP-Ova, serum IgG1/IgG2c, and intestinal IgA antibody titers remained stable. Administration of LPs exerted no adverse effects on immunized mice as no morbidity or signs of toxicity were observed for the duration of the studies. These results indicate that antigen-conjugated liposomes are immunogenic and confirm a previous report that mucosal priming followed by a s.c. boosting immunization is the most effective strategy for inducing long-lasting mucosal IgA, as well as a polarized Th1/Th2 systemic response. In addition to being biodegradable and easily functionalized by conjugation, liposomes have a hollow core which can also be loaded with cargo, allowing for a targeted delivery of multiple antigens (or drugs) simultaneously. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 557-565, 2017.

Polystyrene microspheres enable 10-color compensation for immunophenotyping of primary human leukocytes.

Compensation is a critical process for the unbiased analysis of flow cytometry data. Numerous compensation strategies exist, including the use of bead-based products. The purpose of this study was to determine whether beads, specifically polystyrene microspheres (PSMS) compare to the use of primary leukocytes for single color based compensation when conducting polychromatic flow cytometry. To do so, we stained individual tubes of both PSMS and leukocytes with panel specific antibodies conjugated to fluorochromes corresponding to fluorescent channels FL1-FL10. We compared the matrix generated by PSMS to that generated using peripheral blood mononuclear cells (PBMC). Ideal for compensation is a sample with both a discrete negative population and a bright positive population. We demonstrate that PSMS display autofluorescence properties similar to PBMC. When comparing PSMS to PBMC for compensation PSMS yielded more evenly distributed and discrete negative and positive populations to use for compensation. We analyzed three donors' PBMC stained with our 10-color T cell subpopulation panel using compensation generated by PSMS vs.PBMC and detected no significant differences in the population distribution. Panel specific antibodies bound to PSMS represent an invaluable valid tool to generate suitable compensation matrices especially when sample material is limited and/or the sample requires analysis of dynamically modulated or rare events.

Latex-protein complexes from an acute phase recombinant antigen of Toxoplasma gondii for the diagnosis of recently acquired toxoplasmosis.

The synthesis and characterization of latex-protein complexes (LPC), from the acute phase recombinant antigen P35 (P35Ag) of Toxoplasma gondii and "core-shell" carboxylated or polystyrene (PS) latexes (of different sizes and charge densities) are considered, with the aim of producing immunoagglutination reagents able to detect recently acquired toxoplasmosis. Physical adsorption (PA) and chemical coupling (CC) of P35Ag onto latex particles at different pH were investigated. Greater amounts of adsorbed protein were obtained on PS latexes than on carboxylated latexes, indicating that hydrophobic forces govern the interactions between the protein and the particle surface. In the CC experiments, the highest amount of bound protein was obtained at pH 6, near the isoelectric point of the protein (IP=6.27). At this pH, it decreased both the repulsion between particle surface and protein, and the repulsion between neighboring molecules. The LPC were characterized and the antigenicity of the P35Ag protein coupled on the particles surface was evaluated by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results from ELISA showed that the P35Ag coupled to the latex particles surface was not affected during the particles sensitization by PA and CC and the produced LPC were able to recognize specific anti-P35Ag antibodies present in the acute phase of the disease.

In vitro and ex vivo characterization of lectin-labeled Mycobacterium tuberculosis antigen-containing microspheres for enhanced oral delivery.

PURPOSE: Oral immunization for mucosal protection against Mycobacterium tuberculosis would be the best option for effective tuberculosis (TB) control. However, this route of vaccine delivery is limited due to the short residence time of the delivery system at the site of absorption. Cytoadhension has made it possible to optimize the targeted delivery of oral vaccine to lymphoid tissues. The purpose of this project was to evaluate the ability of human M-cell specific lectin-labeled microparticles to target the human M-cells of the Peyer's patches. METHOD: Albumin microspheres containing Mycobacterium tuberculosis cell lysate antigens were coupled with Wheat germ agglutinin and Aleuria aurantia lectins and their ability to bind to M cell models as well as their preferential distribution in the Peyer's patches were investigated. RESULTS: The study demonstrated an enhanced delivery of targeted polystyrene and BSA/Lysate microspheres to M cells. It was demonstrated that alpha-l-fucose sugar residue might be the target of these lectins. CONCLUSION: It can be concluded from the study that the lectin-coupled microspheres had better affinity for M-cells and showed preferential binding to the Peyer's patches. This means that the coupling enhanced the targeted delivery of the antigens to the M cells.

Methods of effective conjugation of antigens to nanoparticles as non-inflammatory vaccine carriers.

It has recently become clear that nanoparticle size is a major determinant for how antigen presenting cells (APCs), and specifically dendritic cells (DC) recognize and handle particles, and hence a critical parameter for the formulation of particulate vaccines that aim to induce immunity by targeting DC. Our previous studies in mice and sheep have shown polystyrene nanoparticles of 40-50 nm (PSNPs) with covalently bound antigen offer a new class of vaccines, which contain only 2 elements, antigen and particle, and no added inflammatory stimuli, but evoke very potent combined CD8 T cell and antibody responses. Herein we have optimized the methods for antigen conjugation to PSNPs to controllably promote a single antigen (protein or peptide) layer coating on the nanoparticle. Surprisingly, these nanovaccines not only continued to induce high levels of CD8 T cells in vivo, but were further more potent antibody inducers than nanoparticles containing multiple antigen layers. Addressing the issue of antigen loading on PSNPs, we found an optimal range, above or below which immunogenicity is changed either for antibodies or CD8 T cells. The mechanism behind the induction of high levels of CD8 T cells was further explored by assessing the DC subset that takes up the PSNPs in vivo, and these were found to be preferentially CD8(+) CD11c(+) DC in the lymph node draining the injection site. Since the levels of induced antibodies were highly elevated, and CD8(+) DC do not traditionally induce antibodies, we further sought to find if, despite no detectable inflammation at the injection site, the PSNPs may perhaps induce inflammatory cytokines locally in the lymph node after injection, or systemically in sera, resulting in an adjuvant effect. The initial findings presented herein show no detectable induction of the key inflammatory cytokines such as TNF-α, IL-1 or IL-6, suggesting a novel "non-inflammatory" adjuvant mechanism.

Size-dependent uptake of particles by pulmonary antigen-presenting cell populations and trafficking to regional lymph nodes.

The respiratory tract is an attractive target organ for novel diagnostic and therapeutic applications with nano-sized carriers, but their immune effects and interactions with key resident antigen-presenting cells (APCs) such as dendritic cells (DCs) and alveolar macrophages (AMs) in different anatomical compartments remain poorly understood. Polystyrene particles ranging from 20 nm to 1,000 nm were instilled intranasally in BALB/c mice, and their interactions with APC populations in airways, lung parenchyma, and lung-draining lymph nodes (LDLNs) were examined after 2 and 24 hours by flow cytometry and confocal microscopy. In the main conducting airways and lung parenchyma, DC subpopulations preferentially captured 20-nm particles, compared with 1,000-nm particles that were transported to the LDLNs by migratory CD11blow DCs and that were observed in close proximity to CD3⁺ T cells. Generally, the uptake of particles increased the expression of CD40 and CD86 in all DC populations, independent of particle size, whereas 20-nm particles induced enhanced antigen presentation to CD4⁺ T cells in LDLNs in vivo. Despite measurable uptake by DCs, the majority of particles were taken up by AMs, irrespective of size. Confocal microscopy and FACS analysis showed few particles in the main conducting airways, but a homogeneous distribution of all particle sizes was evident in the lung parenchyma, mostly confined to AMs. Particulate size as a key parameter determining uptake and trafficking therefore determines the fate of inhaled particulates, and this may have important consequences in the development of novel carriers for pulmonary diagnostic or therapeutic applications.

The signalling imprints of nanoparticle uptake by bone marrow derived dendritic cells.

Nanoparticles (NP) possess remarkable adjuvant and carrier capacity, therefore are used in the development of various vaccine formulations. Our previous studies demonstrated that inert non-toxic 40-50 nm polystyrene NP (PS-NP) can promote strong CD8 T cell and antibody responses to the antigen, in the absence of observable inflammatory responses. Furthermore, instillation of PS-NP inhibited the development of allergic airway inflammation by induction of an immunological imprint via modulation of dendritic cell (DC) function without inducing oxidative stress in the lungs in mice. This is in contrast to many studies which show that a variety of ambient and man-made NP promote lung immunopathology, raising concerns generally about the safe use of NPs in biomedicine. Most NPs are capable of inducing inflammatory pathways in DC largely mediated by signalling via the extracellular signal-regulated kinase 1/2 (ERK). Herein, we investigate whether PS-NPs also activate ERK in DC in vitro. Our data show that PS-NP do not induce ERK activation in two different types of bone marrow derived (BM) DC cultures (expanded with GM-CSF or with GM-CSF together with IL-4). The absence of such signalling was not due to lack of PS-NP uptake by BM-DC as confirmed by confocal microscopy and flow cytometry. The process of NP uptake by DC usually initiates ERK signalling, suggesting an unusual uptake pathway may be engaged by PS-NPs. Indeed, data herein showns that uptake of PS-NP by BM-DC was substantially inhibited by phorbol myristate acetate (PMA) but not cytochalasin D (CCD), suggesting an uptake pathway utilising caveole for PS-NP. Together these data show that BM-DC take up PS-NP via a caveole-dependent pathway which does not trigger ERK signalling which may explain their efficient uptake by DC, without the concomitant activation of conventional inflammatory pathways.

Improved lectin ELISA for glycosylation analysis of biomarkers using PS-tag-fused single-chain Fv.

In this study, we successfully developed a novel lectin enzyme-linked immunosorbent assay (lectin ELISA) for the detection of glycosides linked to carcinoembryonic antigen (CEA) as a model antigen using a scFv-immobilized hydrophilic polystyrene (phi-PS) plate and 12 different HRP-labeled lectins. Anti-CEA scFv genetically fused with a PS-tag at its C-terminus (scFv-PS) was successfully over-expressed in an insoluble fraction by cultivation of recombinant E. coli. A solid-phase refolding method was adopted to immobilize and refold scFv-PS on the surface of the phi-PS plate. Consequently, in sandwich ELISA using phi-PS plates immobilized with scFv and scFv-PS as well as Maxisorp™ plate with whole antibody (whole Ab), the highest sensitivity and S/N ratio were obtained from the scFv-PS-immobilized phi-PS plate as it was the antigen-binding domain with the highest surface immobilization density and remaining activity displayed on the phi-PS surface. During the lectin ELISA, high background signals were detected from the whole-Ab-immobilized Maxisorp™ plate, indicating that HRP-labeled lectins, particularly PHA-E, Con A, LCA, PSA, DSA, and MAA, directly recognized the glyco-chains of a whole Ab. However, a considerable amount of low background signals were detected from the scFv-PS-immobilized plate since the ligand antibody, namely scFv-PS, was produced by E. coli cells that had no potential for glycosylation during the process of post-transcriptional modification. Signals for glyco-chains conjugated with CEA were detectable using 6 kinds of HRP-labeled lectins, namely PHA-E, PHA-L, SBA, Con A, LCA, and DSA, with much higher sensitivities and S/N ratios. Thus, the lectin ELISA using scFv-PS as a ligand was considerably useful for detecting the glyco-chains of glycoproteins, including glyco-biomarkers, with much higher sensitivities and S/N ratios compared with a conventional whole Ab. By preparation of a phi-PS plate immobilized with different species of scFvs, this method is capable of the glyco-chain analysis of a number of glyco-biomarkers in the fields of clinical diagnosis and biochemical research.

Lipopolysaccharide induces multinuclear cell from RAW264.7 line with increased phagocytosis activity.

Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, induces strong proinflammatory responses, including the release of cytokines and nitric oxide from macrophage. In this study, we found that a murine macrophage-derived line, RAW264.7, became multinuclear through cell-cell fusion after incubation with highly purified LPS or synthetic lipid A in the presence of Ca(2+). The same cell line is known to differentiate into multinuclear osteoclast, which expresses a specific proton pumping ATPase together with osteoclast markers on stimulation by the extracellular domain of receptor activator of nuclear factor κB ligand (Toyomura, T., Murata, Y., Yamamoto, A., Oka, T., Sun-Wada, G.-H., Wada, Y. and Futai, M., 2003). The LPS-induced multinuclear cells did not express osteoclast-specific enzymes including tartrate-resistant acid phosphatase and cathepsin K. During multinuclear cell formation, the cells internalized more and larger polystyrene beads (diameter 6-15 µm) than mononuclear cells and osteoclasts. The internalized beads were located in lysosome-marker positive organelles, which were probably phagolysosomes. The LPS-induced multinuclear cell could be a good model system to study phagocytosis of large foreign bodies.

Antibody-antigen interaction on polystyrene: an in situ ellipsometric study.

The objective of this investigation was to monitor the adsorption of antibodies to polystyrene surfaces using ellipsometry. Commercial polystyrene slides used for solid state diagnostics were selected as substrates and the adsorption of three different antibodies (human IgG, bovine IgG and goat anti-human IgG) were evaluated. Based on theoretical models describing the ellipsometric data, it was concluded that the adsorption of antibodies should result in layers that are sufficiently thick to be able to monitor the adsorption in terms of adsorbed amount and thickness of the layer with a reasonable precision. The experimental results confirmed this assumption and values of 2.0-2.3 mg/m(2) were detected for the adsorbed amount with a corresponding thickness of 10-16 nm. It was furthermore found that the antibodies bound irreversibly with respect to rinsing with protein-free solutions. In additional experiments, the consecutive incubation of human IgG and anti-human IgG was investigated. These results showed that, on average, approximately half of the surface immobilized anti-human IgG molecules are capable of binding to human IgG during its incubation. From the consecutive binding experiments it could also be concluded that antibodies present in the polyclonal anti-human IgG preparation were capable of binding to around four different epitopes on the human IgG. A final set of experiments addressed the stability of adsorbed human IgG layers with respect to drying and incubation with surfactant. The results revealed that the adsorbed antibody layer is relatively resistant to these treatments.

Decreased material-activation of the complement system using low-energy plasma polymerized poly(vinyl pyrrolidone) coatings.

In the current study we investigate the activation of blood complement on medical device silicone rubber and present a plasma polymerized vinyl pyrrolidone (ppVP) coating which strongly decreases surface-activation of the blood complement system. We show that uncoated silicone and polystyrene are both potent activators of the complement system, measured both as activated, deposited C3b and quantifying fluid-phase release of the cleavage fragment C3c. The ppVP coated silicone exhibits approximately 90% reduced complement activation compared to untreated silicone. Quartz crystal microbalance with dissipation (QCM-D) measurements show relatively strong adsorption of blood proteins including native C3 to the ppVP surface, indicating that reduction of complement activation on ppVP is neither a result of low protein adsorption nor lower direct C3-binding, and is therefore possibly a consequence of differences in the adsorbed protein layer composition. The alternative and classical complement pathways are barely detectable on ppVP while the lectin pathway through MBL/ficolin-2 deposition remains active on ppVP suggesting this pathway is responsible for the remaining subtle activation on the ppVP coated surface. The ppVP surface is furthermore characterized physically and chemically using scanning electron microscopy (SEM), x-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR), which indicates preservation of chemical functionality by the applied plasma process. Overall, the ppVP coating shows a potential for increasing complement-compatibility of blood-contacting devices.

Fabrication of anionic sulfate-functionalized nanoparticles as an immunosensor by protein immobilization.

Anionic sulfate (SO(4)(-))-functionalized polystyrene (PS) nanoparticles were prepared by the thermal decomposition of potassium persulfate (KPS) in the presence of sodium tetraborate via emulsion polymerization. The presence of a SO(4)(-) group at a solid/liquid interface of a particle surface was confirmed by a zeta potential value of -40.6 mV as well as the shifting of S 2p spectra toward a lower-binding-energy region around 162.7 eV (2p(3/2)) and 164.4 eV (2p(1/2)) in X-ray photoelectron spectroscopy (XPS) analysis. The electrostatic attraction between positively charged antibodies of human immunoglobulin G (hIgG) and cardiac troponin I (cTnI) and negatively charged particle surfaces was accomplished. The atomic force microscopy (AFM) measurement and bicinchoninic acid (BCA) assay results show binding structure between hIgG and antibodies of hIgG (anti-hIgG) with a gradual increase in particle diameter to 152.6 nm (bare), 170.2 nm (hIgG), and 178.9 nm (hIgG/anti-hIgG). Surface coverage densities of 331.4 ng/cm(2) (hIgG) and 320.3 ng/cm(2) (cTnI) and the binding capacity of hIgG to HyLite-750-labeled Fab-specific anti-hIgG (approximately 81.2%) indicate that the majority of hIgG was immobilized with a Y-shaped orientation. The sandwich immunoassay results provide the evidence that the immunological activity of cTnI on the PS nanoparticle surface was retained because the binding activity of the cTnI-PS nanoparticle/cTnI (antigen)/detection cTnI-antibody reaction showed a 5-fold higher activity than that of the cTnI-PS nanoparticle/human serum albumin (HSA)/detection cTnI antibody used as a negative control.

Targeting very small model lesions pretargeted with bispecific antibody with 99mTc-labeled high-specific radioactivity polymers.

BACKGROUND: Two-step targeting with bispecific antibody and Tc-labeled high-specific radioactivity polymers was used for molecular imaging of two very small model lesions in rats. METHODS AND RESULTS: Sprague-Dawley rats (group I) were injected with surrogate antigen-coated beads (SA beads) in the right hind leg or unmodified beads in the contralateral hind leg. In group II, femoral artery de-endothelialization was induced in the left hind leg and sham operation was performed in the contralateral hind leg. Bispecific antibody Z2D3 F(ab')2-anti-DTPA F(ab')2 was injected intravenously 24 h after SAB injection or 1 week after endothelial denudation. Tc-labeled polymers were injected intravenously 24 h later and gamma-images obtained at 2 and 24 h in group I or approximately 2.5 h in group II. Lesions were visualized by 2 h. In group I, SA beads-specific uptake in muscles was significantly greater than with unmodified beads (P<0.015). In group II, lesions were visualized by 2.5 h after radiopolymer injection with uptake activity 2.1+/-0.6 times greater than in the contralateral side from in-vivo images (P<0.004) and 1.8+/-0.7 times by gamma-scintillation counting (P<0.04). CONCLUSION: Pretargeting with Z2D3 bispecific antibody for the localization of radiolabeled polymers enabled successful in-vivo gamma-imaging of very small lesions in two rat models of extravascular and intravascular targets. Biodistribution data confirmed that pretargeting with bispecific antibody enabled targeted visualization of two different very small model lesions by in-vivo gamma-imaging.

Particle size determines activation of the innate immune system in the lung.

Our knowledge about particle size in relation to activation of the innate immune system is limited. Therefore, the acute effect of particle exposure on the innate immune system was studied in a lung model using the intracellular bacterium Listeria monocytogenes. Female Balb/cA mice were instilled intratracheally with polystyrene particles (PSP) of different diameters (0.064, 0.202, 1.053 and 4.646 mum) simultaneously with or 1 day prior to inoculation of 10(5) bacteria. Mice were sacrificed 1 day after Listeria challenge, and the numbers of viable bacteria in the lungs and the spleen were determined as a measure of cellular activation. In separate experiments, bronchoalveolar lavage (BAL) fluid was collected. Only mice exposed to the smallest PSP (0.064 and 0.202 mum) had significantly reduced bacterial numbers in the lung after particles and Listeria were given simultaneously. When particles were given 1 day prior to Listeria challenge also the largest 4.646 mum PSP, but not the medium size 1.053 mum PSP, reduced bacterial numbers. The number of neutrophils in BAL fluid was increased for all PSP-exposed groups after 24 h, and tended to be highest in the group exposed to 4.646 mum PSP. TNF-alpha, IL-1beta and MIP-2 were significantly increased in BAL fluid after exposure to the largest compared with the smallest PSP. In conclusion, activation of the innate immune system by chemical-free particles was size-dependent. Ultrafine and coarse particles appeared to activate cells by different mechanisms, which implies qualitative differences between the health effects of ambient air particulate matter size fractions.

Induction of potent CD8+ T-cell responses by novel biodegradable nanoparticles carrying human immunodeficiency virus type 1 gp120.

The mainstream of recent anti-AIDS vaccines is a prime/boost approach with multiple doses of the target DNA of human immunodeficiency virus type 1 (HIV-1) and recombinant viral vectors. In this study, we have attempted to construct an efficient protein-based vaccine using biodegradable poly(gamma-glutamic acid) (gamma-PGA) nanoparticles (NPs), which are capable of inducing potent cellular immunity. A significant expansion of CD8+ T cells specific to the major histocompatibility complex class I-restricted gp120 epitope was observed in mice intranasally immunized once with gp120-carrying NPs but not with gp120 alone or gp120 together with the B-subunit of cholera toxin. Both the gp120-encapsulating and -immobilizing forms of NPs could induce antigen-specific spleen CD8+ T cells having a functional profile of cytotoxic T lymphocytes. Long-lived memory CD8+ T cells could also be elicited. Although a substantial decay in the effector memory T cells was observed over time in the immunized mice, the central memory T cells remained relatively constant from day 30 to day 238 after immunization. Furthermore, the memory CD8+ T cells rapidly expanded with boosting with the same immunogen. In addition, gamma-PGA NPs were found to be a much stronger inducer of antigen-specific CD8+ T-cell responses than nonbiodegradable polystyrene NPs. Thus, gamma-PGA NPs carrying various HIV-1 antigens may have great potential as a novel priming and/or boosting tool in current vaccination regimens for the induction of cellular immune responses.

Differential macrophage modulation of asymmetric IgG antibody synthesis by soluble or particulate stimuli.

Asymmetric IgG antibodies behave as antigen-blocking due to the presence of a mannose-rich oligosaccharide residue in one of the Fab fragments of the molecule. This feature prevents the interaction of this antibody with the antigenic epitope, then they are unable to activate of the immune effector mechanisms. Taking into account that macrophages modulate the adaptive immune response according to the nature of the stimulating antigen and that IL-6 increases the synthesis of asymmetric IgG, in this work we analysed the differential modulation of the synthesis of asymmetric IgG by culture supernatants from peritoneal macrophages (CSPM) stimulated with either OVA-polystyrene beads or soluble OVA. The levels of IL-10, IL-6 and TNFalpha were determined in CSPM to find elevated levels of IL-6 and TNFalpha only in those cultures stimulated with OVA-coated polystyrene beads. The 112D5 hybridoma was cultured in the presence of CSPM from cells stimulated with OVA-polystyrene beads or soluble OVA. There was an increase in the synthesis of asymmetric IgG only in those cultures of the hybridoma treated with CSPM stimulated with OVA-polystyrene beads this CSPM also had an antiproliferative effect on the hybridoma. A neutralizing anti-IL-6 antibody inhibited the synthesis of asymmetric IgG but could not revert the effect of such CSPM on the viability of the hybridoma. In addition, our results demonstrated that the murine TNFalpha, at a concentration similar to that found in the particle-stimulated CSPM, had an inhibitory effect on cell growth. In summary, IL-6 and TNFalpha are secreted at different concentrations by peritoneal macrophages depending upon the physicochemical nature of the stimulus, thus conditioning the humoral immune response elicited. The IL-6 present in the CSPM can regulate the secretion of asymmetric IgG and, together with TNFalpha can also regulate cell proliferation. Results obtained in this work would contribute to the knowledge of the role of macrophages in the modulation of the quality of the humoral immune response generated upon stimulation with particulate antigens.

Effects of prosthetic materials on the host immune response: evaluation of polymethyl-methacrylate (PMMA), polyethylene (PE), and polystyrene (PS) particles.

The main causes for the long-term prosthetic implants' failure are the body's reaction to the implanted material or mechanical stress on the device resulting in the formation of wear particles. Particulate wear debris attracts macrophages, and depending on the chemical composition of the material and particle size, various levels of inflammatory response may occur. While transient inflammation is common, development of chronic inflammation may have serious consequences, leading to implant failure. Such a process may also cause systemic changes to immune functions and long-term effects on the host immune responses. In this study, we evaluated the effects of polystyrene (PS), polyethylene (PE), and polymethylmethacrylate (PMMA) particles on macrophage function and the generation of T-cell responses. Particles of various diameters were injected intraperitoneally into Balb/c mice, and immune functions were examined at 3, 10, and 21 days after the injection. The intensity of phagocytosis by peritoneal exudate cells (PECs) and the proliferative response of spleen cells from treated mice were evaluated. Enumeration of PECs revealed an increase in the total number of cells. Mice injected with PS or PE particles had a higher percentage of cells containing particles than PMMA-injected mice. Macrophages with PS or PE particles tended to adhere to and/or infiltrate peritoneal fibro-fatty tissues surrounding the spleen and pancreas, while the PMMA-carrying macrophages infiltrated the spleen, resulting in an increase of spleen size and "weight. The spleen cell proliferation assay revealed only mild and transient effects on the mitogen response in both PE and PS particle-injected mice. However, in the PMMA-injected mice we observed a lasting increase of the Con A response and a decrease of the LPS response. In vitro exposure of PECs from untreated mice showed a dose-response pattern in nitric oxide (NO) and TNFalpha production. While exposure to either PMMA or PE induced comparable levels of NO, exposure to PMMA induced a markedly higher production of TNFalpha than exposure to PE. The results indicate that particulate biomaterials may, in addition to the initial activation of phagocytes, significantly affect immune functions and compromise the host response to other antigenic stimuli.

The IgE adjuvant effect of particles: characterisation of the primary cellular response in the draining lymph node.

Diesel exhaust particles, and polystyrene particles (PSP) as a model for the insoluble particle core, have an adjuvant effect on allergen-specific IgE production in mice. We therefore examined the primary immune response in the draining popliteal lymph node (PLN) to the allergen ovalbumin (OVA) injected together with polystyrene particles into the footpad of BALB/cA mice. Similar numbers of particle-containing cells were observed in the draining lymph node on day 1 after injection of PSP alone or OVA + PSP, the numbers increasing continuously until day 21. The total lymph node cell numbers increased three to four times in the OVA + PSP group compared to both OVA and PSP groups, peaking on day 5. The increase in B cell numbers was twice the increase in T cell numbers. On day 5, OVA + PSP increased the expression of most surface markers measured (MHC class II, CD86, CD23, CD69) compared to OVA and PSP. Further, the ex vivo production of IL-4 and IL-10 by PLN cells from OVA + PSP-injected animals was increased. In conclusion, whereas PSP alone did not influence any of the immunologic markers studied, the adjuvant effect of PSP on the IgE antibody response to OVA was associated with an early increased primary cellular response in the draining lymph node.