Immune-regulated IDO1-dependent tryptophan metabolism is source of one-carbon units for pancreatic cancer and stellate cells.

Cancer cells adapt their metabolism to support elevated energetic and anabolic demands of proliferation. Folate-dependent one-carbon metabolism is a critical metabolic process underpinning cellular proliferation supplying carbons for the synthesis of nucleotides incorporated into DNA and RNA. Recent research has focused on the nutrients that supply one-carbons to the folate cycle, particularly serine. Tryptophan is a theoretical source of one-carbon units through metabolism by IDO1, an enzyme intensively investigated in the context of tumor immune evasion. Using in vitro and in vivo pancreatic cancer models, we show that IDO1 expression is highly context dependent, influenced by attachment-independent growth and the canonical activator IFNγ. In IDO1-expressing cancer cells, tryptophan is a bona fide one-carbon donor for purine nucleotide synthesis in vitro and in vivo. Furthermore, we show that cancer cells release tryptophan-derived formate, which can be used by pancreatic stellate cells to support purine nucleotide synthesis.

Protective effect of catechin on humoral and cell mediated immunity in rat model.

The present study was focused on examining the effect of catechin on the cellular and humoral immunity in rat model. Immunomodulatory effect of catechin was determined by delayed-type hypersensitivity (DTH) response, carbon clearance assay, leucocyte mobilization test and cyclophosphamide-induced myelosuppression and hemagglutinating antibody (HA) titer assay. Catechin in experimental dose (25, 50 and 100mg/kg, p.o.) elevated a significant increase in antibody titer in the hemagglutination test with increased levels of immunoglobulin. There was an enhancement in the delayed-type hypersensitivity reaction produced by sheep red blood cells. There was also restoration in the functioning of leucocytes in cyclophosphamide-treated rats with an increased clearance of carbon particles. The results of the present study signify that catechin possesses sufficient potential for modulating immune activity by cellular and humoral mechanisms.

Simultaneous detection of two tumor markers using silver and gold nanoparticles decorated carbon nanospheres as labels.

In this work, a multiplexed electrochemical immunosensor was developed for sensitive detection of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) using silver nanoparticles (Ag NPs) or gold nanoparticles (Au NPs) coated-carbon nanospheres (CNSs) as labels. CNSs were employed as the carrier for the immobilization of nanoparticles (Ag NPs or Au NPs), thionine (Thi), and secondary antibodies (Ab2) due to their good monodispersity and uniform structure. Au NPs reduced graphene oxide (rGO) nanocomposites were used as sensing substrate for assembling two primary antibodies (Ab1). In the presence of target proteins, two labels were attached onto the surface of the rGO/Au NPs nanocomposites via a sandwich immunoreaction. Two distinguishable peaks, one at +0.16 V (corresponding to Ag NPs) and another at -0.33 V (corresponding to Thi), were obtained in differential pulse voltammetry (DPV). The peak difference was approximately 490 mV, indicating that CEA and AFP can be simultaneously detected in a single run. Under optimal conditions, the peak currents were linearly related to the concentrations of CEA or AFP in the range of 0.01-80 ng ml(-1). The detection limits of CEA and AFP were 2.8 and 3.5 pg ml(-1), respectively (at a signal-to-noise ratio of 3). Moreover, when the immunosensor was applied to serum samples, the results obtained were in agreement with those of the reference method, indicating that the immunosensor would be promising in the application of clinical diagnosis and screening of biomarkers.

Synergistic Costimulatory Effect of Chlamydia pneumoniae with Carbon Nanoparticles on NLRP3 Inflammasome-Mediated Interleukin-1ß Secretion in Macrophages.

The obligate intracellular bacterium Chlamydia pneumoniae is not only a causative agent of community-acquired pneumonia but is also associated with a more serious chronic disease, asthma, which might be exacerbated by air pollution containing carbon nanoparticles. Although a detailed mechanism of exacerbation remains unknown, the proinflammatory cytokine interleukin-1ß (IL-1ß) is a critical player in the pathogenesis of asthma. C. pneumoniae induces IL-1ß in macrophages via NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome activation and Toll-like receptor 2/4 (TLR2/4) stimulation. Carbon nanoparticles, such as carbon nanotubes (CNTs), can also evoke the NLRP3 inflammasome to trigger IL-1ß secretion from lipopolysaccharide-primed macrophages. This study assessed whether costimulation of C. pneumoniae with CNTs synergistically enhanced IL-1ß secretion from macrophages, and determined the molecular mechanism involved. Enhanced IL-1ß secretion from C. pneumoniae-infected macrophages by CNTs was dose and time dependent. Transmission electron microscopy revealed that C. pneumoniae and CNTs were engulfed concurrently by macrophages. Inhibitors of actin polymerization or caspase-1, a component of the inflammasome, significantly blocked IL-1ß secretion. Gene silencing using small interfering RNA (siRNA) targeting the NLRP3 gene also abolished IL-1ß secretion. Other inhibitors (K(+) efflux inhibitor, cathepsin B inhibitor, and reactive oxygen species-generating inhibitor) also blocked IL-1ß secretion. Taken together, these findings demonstrated that CNTs synergistically enhanced IL-1ß secretion from C. pneumoniae-infected macrophages via the NLRP3 inflammasome and caspase-1 activation, providing novel insight into our understanding of how C. pneumoniae infection can exacerbate asthma.

Biomarkers of one-carbon metabolism are associated with biomarkers of inflammation in women.

Folate-mediated one-carbon metabolism is essential for DNA synthesis, repair, and methylation. Perturbations in one-carbon metabolism have been implicated in increased risk of some cancers and may also affect inflammatory processes. We investigated these interrelated pathways to understand their relation. The objective was to explore associations between inflammation and biomarkers of nutritional status and one-carbon metabolism. In a cross-sectional study in 1976 women selected from the Women's Health Initiative Observational Study, plasma vitamin B-6 [pyridoxal-5'-phosphate (PLP)], plasma vitamin B-12, plasma folate, and RBC folate were measured as nutritional biomarkers; serum C-reactive protein (CRP) and serum amyloid A (SAA) were measured as biomarkers of inflammation; and homocysteine and cysteine were measured as integrated biomarkers of one-carbon metabolism. Student's t, chi-square, and Spearman rank correlations, along with multiple linear regressions, were used to explore relations between biomarkers; additionally, we tested stratification by folic acid fortification period and multivitamin use. With the use of univariate analysis, plasma PLP was the only nutritional biomarker that was modestly significantly correlated with serum CRP and SAA (ρ = -0.22 and -0.12, respectively; P < 0.0001). Homocysteine (µmol/L) showed significant inverse correlations with all nutritional biomarkers (ranging from ρ = -0.30 to ρ = -0.46; all P < 0.0001). With the use of multiple linear regression, plasma PLP, RBC folate, homocysteine, and cysteine were identified as independent predictors of CRP; and PLP, vitamin B-12, RBC folate, and homocysteine were identified as predictors of SAA. When stratified by folic acid fortification period, nutrition-homocysteine correlations were generally weaker in the postfortification period, whereas associations between plasma PLP and serum CRP increased. Biomarkers of inflammation are associated with PLP, RBC folate, and homocysteine in women. The connection between the pathways needs to be further investigated and causality established. The trial is registered at clinicaltrials.gov as NCT00000611.

Carbon in airway macrophages from children with asthma.

BACKGROUND: Airway macrophage (AM) phagocytosis is impaired in severe asthma. Prostaglandin (PG) E2 and D2 are increased in severe asthma and suppress AM phagocytic function in vitro. In this study, we sought evidence for PG-mediated impairment of phagocytosis of inhalable carbonaceous particulate matter (PM) by AM in children with severe asthma compared with mild asthmatics and healthy controls. METHODS: AM were obtained from children with asthma and healthy controls using induced sputum. AM carbon area (µm(2)) was assessed by image analysis. In a subgroup of asthmatics, urinary PGE2 and PGD2 metabolites were measured by high-performance liquid chromatography, and PM exposure at the home address was modelled. Phagocytosis of PM by human monocyte-derived macrophages and rat AM was assessed in vitro by image analysis. RESULTS: AM carbon was 51% lower in children with moderate-to-severe asthma (n=36) compared with mild asthmatics (n=12, p<0.01) and healthy controls (n=47, p<0.01). There was no association between modelled PM exposure and AM carbon in 33 asthmatics who had a urine sample, but there was an inverse association between AM carbon and urinary metabolites of PGE2 and D2 (n=33, rs=-0.40, p<0.05, and rs=-0.44, p<0.01). PGE2 10(-6) M, but not PGD2 10(-6) M, suppressed phagocytosis of PM10 by human macrophages in vitro (p<0.05 vs control). PGE2 10(-6) M also suppressed phagocytosis of PM10 by rat AM in vitro (p<0.01 vs control). CONCLUSIONS: Phagocytosis of inhaled carbonaceous PM by AMs is impaired in severe asthma. PGE2 may contribute to impaired AM phagocytic function in severe asthma.

Carbon nanofibers have IgE adjuvant capacity but are less potent than nanotubes in promoting allergic airway responses.

There is a growing concern for the possible health impact of nanoparticles. The main objective of this study was to investigate the allergy-promoting capacity of four different carbon nanofiber (CNF) samples in an injection and an airway mouse model of allergy. Secondly, the potency of the CNF was compared to the previously reported allergy-promoting capacity of carbon nanotubes (CNT) in the airway model. Ultrafine carbon black particles (ufCBP) were used as a positive control. Particles were given together with the allergen ovalbumin (OVA) either by subcutaneous injection into the footpad or intranasally to BALB/cA mice. After allergen booster, OVA-specific IgE, IgG1, and IgG2a in serum were measured. In the airway model, inflammation was determined as influx of inflammatory cells (eosinophils, neutrophils, lymphocytes, and macrophages) and by mediators (MCP-1 and TNF-α present in bronchoalveolar fluid (BALF)). CNF and CNT both increased OVA-specific IgE levels in the two models, but in the airway model, the CNT gave a significantly stronger IgE response than the CNF. Furthermore, the CNT and not the CNF promoted eosinophil lung inflammation. Our data therefore suggest that nanotube-associated properties are particularly potent in promoting allergic responses.

Growth of Candida albicans cells on the physiologically relevant carbon source lactate affects their recognition and phagocytosis by immune cells.

Candida albicans is a normal resident of the human gastrointestinal and urogenital tracts and also a prevalent fungal pathogen. During both commensalism and infection, it must match the immunological defenses of its host while adapting to environmental cues and the local nutrient status. C. albicans regularly colonizes glucose-poor niches, thereby depending upon alternative carbon sources for growth. However, most studies of host immune responses to C. albicans have been performed on fungal cells grown on glucose, and the extent to which alternative physiologically relevant carbon sources impact innate immune responses has not been studied. The fungal cell wall is decorated with multifarious pathogen-associated molecular patterns and is the main target for recognition by host innate immune cells. Cell wall architecture is both robust and dynamic, and it is dramatically influenced by growth conditions. We found that growth of C. albicans cells on lactate, a nonfermentative carbon source available in numerous anatomical niches, modulates their interactions with immune cells and the resultant cytokine profile. Notably, lactate-grown C. albicans stimulated interleukin-10 (IL-10) production while decreasing IL-17 levels, rendering these cells less visible to the immune system than were glucose-grown cells. This trend was observed in clinical C. albicans isolates from different host niches and from different epidemiological clades. In addition, lactate-grown C. albicans cells were taken up by macrophages less efficiently, but they were more efficient at killing and escaping these phagocytic cells. Our data indicate that carbon source has a major impact upon the C. albicans interaction with the innate immune system.

Iron core/shell nanoparticles as magnetic drug carriers: possible interactions with the vascular compartment.

AIMS: Nanomagnets with metal cores have recently been shown to be promising candidates for magnetic drug delivery due to higher magnetic moments compared with commonly used metal oxides. Successful application strongly relies on a safe implementation that goes along with detailed knowledge of interactions and effects that nanomagnets might impart once entering the body. MATERIALS & METHODS: In this work, we put a particular focus on the interactions of ultra-strong metal nanomagnets (≥ three-times higher in magnetization compared with oxide nanoparticles) within the vascular compartment. Individual aspects of possible effects are addressed, including interactions with the coagulation cascade, the complement system, phagocytes and toxic or inflammatory reactions both by blood and endothelial cells in response to nanomagnet exposure. RESULTS: We show that carbon-coated metal nanomagnets are well-tolerated by cells of the vascular compartment and have only minor effects on blood coagulation. CONCLUSION: These findings provide the fundament to initiate successful first in vivo evaluations opening metal nanomagnets with improved magnetic properties to fascinating applications in nanomedicine.

The soluble nickel component of residual oil fly ash alters pulmonary host defense in rats.

The soluble metal fraction of residual oil fly ash (ROFA) has been shown to increase the susceptibility to infection in animal models. The goal of this study was to determine which of the primary soluble metals or metal combinations in ROFA were responsible for the increased infectivity. The soluble fraction of ROFA contained Ni, Fe, Al, and Zn. On Day 0, Sprague-Dawley rats were intratracheally (IT) instilled with NiCl2 (55.7 microg/rat), FeSO4 (32.7 microg/rat), Al3(SO4)2 (46.6 microg/rat), or ZnCl2 (8.69 microg/rat), or a combination of all the metals (Total Mixture). In a separate experiment, rats were instilled with metal mixtures, including the total mixture, and mixtures without Fe (Mix--No Fe), Ni (Mix--No Ni), Al (Mix--No Al), or Zn (Mix--No Zn). At Day 3, rats were instilled with 5 x 10(4) Listeria monocytogenes. At Days 6, 8 and 10, left lungs were removed to assess bacterial clearance. Bronchoalveolar lavage (BAL) was performed on right lungs on Day 3, before infection, and on Days 6, 8 and 10 to assess lung injury and cellular activity. Prior to infection, soluble Ni and mixtures containing Ni significantly increased lung injury, inflammation, and oxidative damage to a comparable degree when compared to control. Post-infection, rats pre-treated with soluble Ni, alone or in a metal mixture, had increased bacterial lung burden on Day 6, and body weight decreased in the soluble Ni, Mix--No Fe, and Mix--No Al groups post-infection, indicating Fe and Al may act antagonistically to Ni. Ni alone and in metal mixtures increased reactive oxidants in the lung and appeared to be the most important factor in suppressing T-cell activity post-infection. Soluble Ni is likely the primary metal involved in the increased susceptibility to infection observed in rats exposed to the soluble metals of ROFA.

Development of an electrochemical immunoassay for rapid detection of E. coli using anodic stripping voltammetry based on Cu@Au nanoparticles as antibody labels.

A sensitive electrochemical immunoassay for rapid detection of Escherichia coli has been developed by anodic stripping voltammetry (ASV) based on core-shell Cu@Au nanoparticles (NPs) as anti-E. coli antibody labels. The characteristics of Cu@Au NPs before and after binding with antibody were confirmed by transmission electron microscopy (TEM). After Cu@Au-labeled antibody reacted with the immobilized E. coli on Polystyrene (PS)-modified ITO chip, Cu@Au NPs were dissolved by oxidation to the metal ionic forms, and the released Cu(2+) ions were determined at GC/Nafion/Hg modified electrode by ASV. The utilization of GC/Nafion/Hg modified electrode could enhance the sensitivity for Cu(2+) detection with a concentration as low as 9.0 x 10(-12)mol/L. Since Cu@Au NPs labels were only present when antibody reacted with E. coli, the amount of Cu(2+) directly reflected the number of E. coli. The technique could detect E. coli with a detection limit of 30CFU/mL and the overall analysis could be completed in 2h. By introducing a pre-enrichment step, a concentration of 3CFU/10mL E. coli in surface water was detected by the electrochemical immunoassay.

Complement activation by PEGylated single-walled carbon nanotubes is independent of C1q and alternative pathway turnover.

We have investigated the interaction between long circulating poly(ethylene glycol)-stabilized single-walled carbon nanotubes (SWNTs) and the complement system. Aminopoly(ethylene glycol)(5000)-distearoylphosphatidylethanolamine (aminoPEG(5000)-DSPE) and methoxyPEG(5000)-DSPE coated as-grown HIPco SWNTs activated complement in undiluted normal human serum as reflected in significant rises in C4d and SC5b-9 levels, but not the alternative pathway split-product Bb, thus indicating activation exclusively through C4 cleavage. Studies in C2-depleted serum confirmed that PEGylated nanotube-mediated elevation of SC5b-9 was C4b2a convertase-dependent. With the aid of monoclonal antibodies against C1s and human serum depleted from C1q, nanotube-mediated complement activation in C1q-depleted serum was also shown to be independent of classical pathway. Nanotube-mediated C4d elevation in C1q-depleted serum, however, was inhibited by N-acetylglucosamine, Futhan (a broad-spectrum serine protease inhibitor capable of preventing complement activation through all three pathways) and anti-MASP-2 antibodies; this strongly suggests a role for activation of MASP-2 in subsequent C4 cleavage and assembly of C4b2a covertases. Intravenous injection of PEGylated nanotubes in some rats was associated with a significant rise in plasma thromboxane B2 levels, indicative of in vivo nanotube-mediated complement activation. The clinical implications of these observations are discussed.

Soluble metals in residual oil fly ash alter innate and adaptive pulmonary immune responses to bacterial infection in rats.

The soluble metals of the pollutant, residual oil fly ash (ROFA), have been shown to alter pulmonary bacterial clearance in rats. The goal of this study was to determine the potential effects on both the innate and adaptive lung immune responses after bacterial infection in rats pre-exposed to the soluble metals in ROFA. Sprague-Dawley rats were intratracheally dosed (i.t.) at day 0 with ROFA (R-Total) (1.0 mg/100 g body weight), the soluble fraction of ROFA (R-Soluble), the soluble sample subject to a chelator (R-Chelex), or phosphate-buffered saline (Saline). On day 3, rats were administered an i.t. dose of 5 x 10(4)Listeria monocytogenes. On days 6, 8, and 10, bacterial pulmonary clearance was monitored and bronchoalveolar lavage (BAL) was performed on days 3 (pre-infection), 6, 8, and 10. A concentrated first fraction of lavage fluid was retained for analysis of lactate dehydrogenase and albumin to assess lung injury. BAL cell number, phenotype, and production of reactive oxygen (ROS) and nitrogen species (RNS) were assessed, and a variety of cytokines were measured in the BAL fluid. Rats pre-treated with R-Soluble showed elevated lung injury/cytotoxicity and increased cellular influx into the lungs. R-Soluble-treatment also altered ROS, RNS, and cytokine levels, and caused a degree of macrophage and T cell inhibition. These effects of R-Soluble result in increased pulmonary bacterial burden after infection. The results suggest that soluble metals in ROFA increase lung injury and inflammation, and alter both innate and adaptive pulmonary immune responses.

Modulation of lymphocyte migration to the murine spleen after marginal zone macrophage phagocytosis of blood-borne particulate material.

Our previous studies identified a role for MZM in the movement of lymphocytes into the splenic white pulp. Here we show that phagocytosis of colloidal carbon by marginal zone macrophages results in a splenic influx of B lymphocytes, and T lymphocytes of memory/activated phenotype, with concomitant upregulation of B Lymphocyte Chemoattractant (BLC, CXCL13) mRNA, a chemokine acting on B and memory/activated T lymphocytes. The recruitment of B cells and activated T cells to the spleen after phagocytic uptake would allow an immune response against blood-borne pathogens to be quickly and effectively mounted by bringing together the two key cell types responsible for generating humoral immunity.

Immunization with soot from a non-combustion process provokes formation of antibodies against polycyclic aromatic hydrocarbons.

The emission of soot during combustion processes used in transportation, manufacturing, and power generation is of increasing concern because of its serious adverse health effects. In particular, the ability to modulate the immune system has recently been established. In the present investigation, an artificial soot sample that was prepared by fragmentation of acetylene in a laser-induced plasma was used as an antigen for the immunization of a rabbit. A highly sensitive competition curve in an indirect competitive ELISA using a benzo[a]pyrene-BSA conjugate as a coating antigen could be constructed for benzo[a]pyrene with an IC50 of 2.94 mug/l (11.65 nmol/l). In contrast to the high affinity, the soot antiserum dilution (antibody titer) of 1:750 was rather low. The cross-reactivity was tested with 16 parent polycyclic aromatic hydrocarbons, 7 nitrated polycyclic aromatics, and 3 methylated, hydroxylated or butyric acid derivatives. The results obtained suggest that the vertebrate immune system can respond to an immunization with soot by the generation of high affinity IgG class antibodies against polycyclic aromatics. It is likely that antibodies are raised against the molecular structures which form the framework of the soot particles and not against adsorbed and extractable polyaromatic compounds. The experiments suggest that if soot is considered a T-independent antigen, the isotype switch, essentially from IgM to IgG, could have been caused by co-inoculation with a T-dependent antigen, i.e., mycobacteria contained in Freunds complete adjuvant. However, at the cellular level the mechanism remains to be uncovered.

[Agglutination and phagocytosis of foreign abiotic particles by hemocytes of the blowely, Calliphora vicina in vivo. I. Dynamics of hemocyte activity during larval development].

Three types of Calliphora larval hemocytes have been revealed to be involved in phagocytosis of abiotic foreign particles: thrombocytoids, larval plasmatocytes and plasmatocytes I. Thrombocytoids are the quickest to respond to the appearance of invaders. The onset of test particle entrapment by thrombocytoid cytoplasmic fragments was observed, depending on the larval age within 0.5-5.0 min after injection. Separated fragments were fused, forming strands or roundish agglutinates. Phagocytosis of carbon, carmine or Indian ink particles by larval plasmatocytes occurs far more lately, and no earlier than 20-30 min after injection. Plasmatocytes I are capable of foreign particles adhesion on their surface, with a subsequent morule formation, and of engulfing these particles. These two events start in different time periods: adhesion occurs in 5-10 min, while phagocytosis is observed in 1--3 h. The rate of test particle entrapment and stability of agglutinales clearly depends on the larval age. The most pronounced reaction of hemocytes to foreign particles may be observed by the end of feeding and crop emptying. The second, somewhat less expressed rise of activity occurs in mature larvae not long before the onset of pupariation. Diapause induction is accompanied by reducing activities of both plasmatocytes and thrompocytoids. The importance of different hemocyte types in cellular immune reaction of Calliphora vicina larvae, and co-ordination between plasmatocytes and thrombocytoids are discussed.

Calcium and ROS-mediated activation of transcription factors and TNF-alpha cytokine gene expression in macrophages exposed to ultrafine particles.

Ultrafine (Uf) particles are a component of particulate air pollution suggested to be responsible for the health effects associated with elevations of this pollutant. We have previously suggested that Uf particles, through the induction of oxidative stress, may induce inflammation in the lung, thus exacerbating preexisting illness in susceptible individuals. Alveolar macrophages are considered to play a key role in particlemediated inflammation and lung disease. The effect of Uf particles on rat alveolar macrophages and human blood monocytes was investigated with reference to the roles of calcium and reactive oxygen species (ROS). TNF-alpha protein release, intracellular calcium concentration, TNF-alpha mRNA expression, and transcription factor activation were studied as end points after treatment of rat alveolar macrophages or peripheral blood monocytes. The calcium channel blocker verapamil, the intracellular calcium chelator BAPTA-AM, the calmodulin inhibitor W-7, and the antioxidants Trolox and Nacystelin (NAL) were included in combination with Uf particles. Verapamil reduced intracellular calcium concentration in rat alveolar macrophages on stimulation with Uf particles. This effect was also apparent with transcription factor AP-1 activation. All antagonists and antioxidants reduced Uf-stimulated nuclear localization of the p50 and p65 subunits of NF-kappaB in human monocytes. Verapamil, BAPTA-AM, and NAL reduced Uf-stimulated TNF-alpha protein release, whereas only verapamil reduced Uf-stimulated mRNA expression in rat alveolar macrophages. In human monocytes, verapamil, Trolox, BAPTA-AM, and W-7 reduced Uf-stimulated TNF-alpha protein release. These findings suggest that Uf particles may exert proinflammatory effects by modulating intracellular calcium concentrations, activation of transcription factors, and cytokine production through a ROS-mediated mechanism.

The effect of diesel exhaust particles (DEP) and carbon black (CB) on thiol changes in pulmonary ovalbumin allergic sensitized Brown Norway rats.

Brown Norway rats were exposed by intratracheal instillation of saline, carbon black (CB), or diesel exhaust particles (DEP) (5 mg/kg) on day 1, followed by exposure to ovalbumin (OVA, 90 mg/m(3)) or saline for 30 minutes on days 1, 8, 15, and 29. Animals were sacrificed on day 30. The DEP, CB, or OVA exposure alone did not result in abnormal levels of inflammatory cells, lactate dehydrogenase (LDH), or total protein in the lavage fluid. In combined OVA-DEP or OVA-CB exposure, however, these markers were significantly increased. The adjuvant effect of CB and DEP on OVA sensitization was evidenced by the marked increases in serum OVA-specific IgG (5.6-fold) and IgE (3.5-4 fold) levels, and the increase in interleukin-4 (IL-4) mRNA levels in lung tissue. The OVA exposure markedly reduced glutathione (GSH) levels in both cell types. In combined DEP-OVA exposure, the level of GSH in lymphocytes was further decreased, indicating a possible interactive effect between DEP and OVA exposures. These results show that both DEP and CB augmented OVA-induced allergic sensitization, and that particle composition of DEP may not be a critical factor for the adjuvant effect. OVA exposure causes significant depletion of intracellular GSH in lymphocytes, which may play a key role in OVA-mediated immune responses.

Molecular dynamics analysis of a buckyball-antibody complex.

This is a multinanosecond molecular dynamics study of a bio-nano complex formed by a carbon nanoparticle, a buckyball C(60), and a biological molecule, an antibody, with high binding affinity and specificity. In the simulation, the ball is completely desolvated by the binding site of the antibody by means of a nearly perfect shape complementarity and extensive side-chain interactions, with the exception that about 17% of the surface is persistently exposed to solvent and could be used for functional derivatization. The interactions are predominantly hydrophobic, but significant polar interactions occur as well. There exists a rich body of various pi-stacking interactions. Aromatic side chains are involved in both double and triple stackings with the ball. Some ionic side chains, such as the guanidinium group of arginine, also form pi-stackings with the ball. The results suggest that pi-stackings are very efficient and common modes of biological recognition of pi-electron-rich carbon nanoparticles. Most importantly, the results demonstrate that, in general, an ordinary protein binding site, such as that of an antibody, can readily bind to a carbon nanoparticle with high affinity and specificity through recognition modes that are common in protein-ligand recognition.

Coadministration of antigen and particles optimally stimulates the immune response in an intranasal administration model in mice.

Some particulate matter is known to affect human health, yet the mechanism(s) by which it acts is largely unknown. One of the factors that may play a role in the immune- stimulating activity of particles is binding of allergen to particles. This may turn the particles into allergen carriers, resulting in antigen deposition within the altered inflammatory microenvironment created by the particles. We compared the efficacy of simultaneous versus separate administration of antigen and particles during sensitization in an intranasal exposure model in BALB/c mice. Sensitization consisted of three separate doses (10 microg) of TNP-OVA at Days 1, 2, and 3. Two hundred micrograms of carbon black particles (CBP) were administered either 1 day before sensitization (Day 0), 1 day after sensitization (Day 4), or during sensitization. The latter was performed either at Day 1 (200 microg) or at Days 1, 2, and 3 (67 microg/day). At Day 10 a challenge with 10 microg of TNP-OVA was performed, and at Day 15 the immune response was assessed. The total number of cells as well as antibody-forming cells (AFC) in lymph nodes draining the lung (peribronchial lymph nodes [PBLN]) were determined, and immunoglobulin levels in blood were assessed. Cell numbers of PBLN increased significantly in all particle-treated groups compared to controls. The number of TNP-specific IgG1-forming cells in the groups receiving particles during sensitization was significantly higher than control level. Only groups receiving particles during or before sensitization displayed significantly higher IgG1 levels than controls, in contrast to the group receiving particles after sensitization. Only in animals receiving three doses of 67 microg during sensitization did TNP-specific IgE increase significantly compared to controls. IgG2a did not show significant differences compared to controls, indicating that the response is predominantly Th2 mediated. These data indicate that coadministration of particles at all time points of antigen dosing is the most effective way to stimulate an immune response in our model compared to separate particle and antigen dosing. Also, administration shortly before antigen administration was effective in stimulating an immune response, suggesting that time-dependent processes are involved in immune-stimulating activity of particles, supporting the important role of the altered inflammatory microenvironment created by the particles.