Antigen presenting cells targeting and stimulation potential of lipoteichoic acid functionalized lipo-polymerosome: a chemo-immunotherapeutic approach against intracellular infectious disease.

Antigen presenting cells (APC) are well-recognized therapeutic targets for intracellular infectious diseases, including visceral leishmaniasis. These targets have raised concerns regarding their potential for drug delivery due to overexpression of a variety of receptors for pathogen associated molecular pathways after infection. Since, lipoteichoic acid (LTA), a surface glycolipid of Gram-positive bacteria responsible for recognition of bacteria by APC receptors that also regulate their activation for pro-inflammatory cytokine secretion, provides additive and significant protection against parasite. Here, we report the nanoarchitechture of APC focused LTA functionalized amphotericin B encapsulated lipo-polymerosome (LTA-AmB-L-Psome) delivery system mediated by self-assembly of synthesized glycol chitosan-stearic acid copolymer (GC-SA) and cholesterol lipid, which can activate and target the chemotherapeutic agents to Leishmania parasite resident APC. Greater J774A and RAW264.7 macrophage internalization of FITC tagged LTA-AmB-L-Psome compared to core AmB-L-Psome was observed by FACSCalibur cytometer assessment. This was further confirmed by higher accumulation in macrophage rich liver, lung and spleen during biodistribution study. The LTA-AmB-L-Psome overcame encapsulated drug toxicity and significantly increased parasite growth inhibition beyond commercial AmB treatment in both in vitro (macrophage-amastigote system; IC50, 0.082 ± 0.009 µg/mL) and in vivo (Leishmania donovani infected hamsters; 89.25 ± 6.44% parasite inhibition) models. Moreover, LTA-AmB-L-Psome stimulated the production of protective cytokines like interferon-γ (IFN-γ), interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase and nitric oxide with down-regulation of disease susceptible cytokines, like transforming growth factor-ß (TGF-ß), IL-10, and IL-4. These data demonstrate the potential use of LTA-functionalized lipo-polymerosome as a biocompatible lucrative nanotherapeutic platform for overcoming toxicity and improving drug efficacy along with induction of robust APC immune responses for effective therapeutics of intracellular diseases.

Prolyl hydroxylases positively regulated LPS-induced inflammation in human gingival fibroblasts via TLR4/MyD88-mediated AKT/NF-κB and MAPK pathways.

OBJECTIVES: Prolyl hydroxylases (PHDs) play essential roles in oxygen-sensing system, whereas the effects of PHDs on inflammation have not been totally uncovered. Our study aimed to investigate the role of PHDs in lipopolysaccharide (LPS)-induced inflammation of human gingival fibroblasts (HGFs) and clarify the potential mechanisms. MATERIALS AND METHODS: A pan hydroxylase inhibitor, dimethyloxallyl glycine (DMOG), and RNA interference were used to explore the role of PHDs in inflammation. Cytotoxic effect of DMOG was determined by cell-counting kit-8 and flow cytometry respectively. The secretion levels of IL-6 and IL-8 were assessed by ELISA. The mRNA levels of inflammatory cytokines, Toll-like receptor (TLR) 4 and MyD88 were evaluated by quantitative real-time PCR. The activation of NF-κB, mitogen-activated protein kinase (MAPK) and PI3K/AKT pathways were detected by western blot and the nuclear translocation of NF-κB p65 was examined by immunofluorescence. Downregulation of PHD1 and PHD2 was performed with siRNA transfection. RESULTS: Dimethyloxallyl glycine inhibited LPS-induced inflammatory cytokine, TLR4 and MyD88 expression in gene level and the elevated secretion of IL-6 and IL-8 was also downregulated. Additionally, LPS-induced activation of NF-κB, MAPK and AKT pathways was abolished by DMOG treatment. Importantly, LPS-induced inflammatory cytokine expression was merely suppressed by PHD2 knockdown. CONCLUSIONS: Prolyl hydroxylases acted as a positive regulator in LPS-induced inflammation of HGFs via TLR4/MyD88-mediated NF-κB, MAPK and AKT signalling pathways and PHD2 among three isoforms was principally responsible for the effects.

Controlled release of lipopolysaccharide in the subarachnoid space of rabbits induces chronic vasospasm in the absence of blood.

OBJECTIVE: Leukocyte-endothelial cell interactions appear to play a role in the development of vasospasm after SAH. Using a purely inflammatory protein, LPS, we evaluated the effect of inflammation on the development of chronic vasospasm in the absence of blood and compared it to SAH-induced vasospasm in rabbits. METHODS: Lipopolysaccharide was incorporated into EVAc polymers to produce 20% LPS/EVAc polymers (wt/wt). Rabbits (n = 23) were randomized to 4 experimental groups: (1) empty polymer (n = 6), (2) SAH (n = 5), (3) 0.7 mg/kg polymeric LPS dose (n = 6), and (4) 1.4 mg/kg polymeric LPS dose (n = 6). Blood and polymers were inserted into the cisterna magna. The rabbits were killed 3 days postoperatively, and the basilar arteries were harvested for morphometric analysis. Clinical response and lumen patencies were analyzed using ANOVA and a post hoc Newman-Keuls Multiple Comparisons test. RESULTS: Significant narrowing of the basilar artery was observed by insertion of 20% LPS/EVAc polymers into the subarachnoid space at a polymeric dose of 1.4 mg/kg (actual dose, 66 microg kg(-1) d(-1)) (75.4% +/- 4.2%; P < .01) and by SAH (80.3% +/- 8.1%; P < .01) as compared with the empty polymer group. A trend toward narrowing was observed in the 0.7 mg/kg polymeric LPS dose group (actual dose, 33 microg kg(-1) d(-1)) (85.2% +/- 2.6%; P > .05). Symptoms associated with SAH were noted in 50% of the rabbits in the 0.7 mg/kg LPS group and in 100% of rabbits in the 1.4 mg/kg LPS group. CONCLUSION: Controlled release of LPS into the subarachnoid space of rabbits produced chronic vasospasm in a dose-dependent manner. At a polymeric dose of 1.4 mg/kg, LPS-induced vasospasm was equivalent to that induced by SAH. This suggests that LPS and SAH may induce vasospasm through similar mechanisms and provides further evidence that inflammation plays a central role in the etiology of chronic vasospasm.

Outward fluid flow reduces inward diffusion of bacterial lipopolysaccharide across intact and demineralised dentine.

OBJECTIVE: To examine the ability of outward fluid flow (OF) on resisting the inward diffusion of bacterial lipopolysaccharide (LPS) across the demineralised dentine (DD) in comparison with that across the intact dentine (ID). DESIGN: Twenty ID discs were prepared from freshly extracted human third molars. After etching both dentine surfaces, hydraulic conductance (L(p)) of the dentine was measured. Ten dentine discs were then completely demineralised using 10% EDTA, and L(p) was re-measured. The diffusion of LPS through ID and DD was measured against the OF and compared to the non-outward flow (NF) (n = 5 for each group) at 0, 1, 4 and 8h. Longitudinal sections of ID and DD surfaces were observed under a scanning electron microscope (SEM). RESULTS: The L(p) of DD was significantly higher than that of ID (independent t-test, p < 0.001). The application of OF and demineralisation significantly affected LPS diffusion (two-way ANOVA, p < 0.05). In addition, the effect of OF depended on dentine demineralisation. SEM images of ID showed intact dentinal tubules, whereas those of DD showed expanded collagen fibres and enlarged dentinal tubules. CONCLUSIONS: The inward diffusion of LPS across DD differed from that of ID and the OF affected the inward diffusion of LPS. In the presence of the OF, the inward diffusion of LPS was reduced to near zero in both ID and DD. Nevertheless, when compared to that in the ID group, the OF produced the slightly greater effect to resist the inward LPS diffusion in the DD group.

Periodontal disease as a risk factor in pregnancy.

There is growing evidence that periodontal disease represents a risk factor for preterm delivery and premature membrane rupture. To prevent an oral inflammatory process evolving into a full-blown form of periodontitis, with possible severe repercussions on pregnancy outcome following systemic spread, future mothers should receive regular professional oral hygiene care throughout pregnancy.

In vivo distribution of particulate antigens and liposomes in murine spleen. A possible role in the humoral immune response.

Several particulate antigens and liposomes were intravenously injected in mice in order to study their localization patterns in spleen and liver. Liposomes have been proposed as promising carriers for haptens and antigens. It was studied whether the phospholipid composition, cholesterol content and charge of the liposomes played a role in their distribution within the spleen. Different thymus-independent type 1 and type 2 and thymus-dependent particulate antigens as well as liposomes were labeled with the lipophilic fluorochrome Di-I. After labeling they were intravenously injected and spleens and livers were removed at different time intervals and prepared for light- and fluorescence-microscopy. We have observed that all particulate antigens and liposomes administered to the mice localized according to the same distribution pattern in the spleen. After 2 and 4 h particles were located in macrophages of the marginal zone and after 24 h white pulp macrophages had also ingested particulate antigens and liposomes. So we conclude that the distribution of the particulate antigens and liposomes in the spleen is independent of the immunological nature of the particles. Results are discussed with respect to the question whether or not the distribution of particulate antigens and liposome associated antigens or haptens, may be a crucial factor in determining the type of immune response to be elicited.

Differences in the permeability of high-flux dialyzer membranes for bacterial pyrogens.

AIMS: The increasing use of high-flux membranes for hemodialysis has raised concerns that patients dialyzed with these membranes may be at higher risk of being exposed to cytokine-inducing bacterial substances in the dialysate than patients dialyzed with low-flux membranes. We investigated the permeability of various high-flux membranes for both purified E. coli lipopolysaccharide (LPS) as well as for LPS derived from Stenotrophomonas (Sten.) maltophilia. MATERIALS AND METHODS: An in vitro dialysis circuit with saline in the blood compartment of 3 dialyzers containing different membranes (polysulfone, helixone and Diapes) was employed. The dialysate was challenged with increasing doses of sterile filtrates derived from Sten. maltophilia cultures or with purified LPS from E. coli. Samples from the blood compartment were tested for cytokine induction (IL-1beta, IL-6 and TNF) in mononuclear cells as well as for LPS by limulus amebocyte lysate test (LAL). RESULTS: IL-6 induction above sterile controls (< 0.02 ng/ml IL-6) was observed by samples from the blood side of DIAPES dialyzers (1.2 +/- 0.7 ng/ml IL-6) after challenging the dialysate with 4.1 +/- 3.6 U/ml E. coli LPS (9.9 +/- 4.5 ng/ml IL-6). In contrast, at the same challenge dose no significant IL-6 induction above sterile controls was observed by blood side samples of polysulfone (0.15 +/- 0.07 ng/ml) and helixone (0.09 +/- 0.05 ng/ml) dialyzers. Increasing the amount of E. coli LPS in the dialysate further augmented IL-6 induction by blood side samples of Diapes but not of polysulfone and helixone dialyzers. Similar results were obtained for IL-1beta and TNF. After challenging the dialysate with E. coli LPS as well as with cultures of Sten. maltophilia, significantly more LAL reactivity was observed in the blood compartment of Diapes compared to polysulfone and helixone. CONCLUSIONS: There are considerable differences between high-flux membranes regarding their permeability for cytokine-inducing substances from E. coli as well as for LPS derived from E. coli and Sten. maltophilia. Dialyzers that leak CIS under aqueous conditions in vivo should not be used unless the dialysate has passed through an ultrafilter.

Ability of bacterial endotoxin to diffuse through human dentin.

An in vitro system was developed to determine whether bacterial endotoxin is capable of diffusing through dentin without the use of filtration pressure. Cavities were prepared in five third molar teeth in order to produce a split chamber device consisting of occlusal and pulpal chambers with 0.5 mm of intervening dentin. Endotoxin was introduced into the occlusal chamber and the effluent in the pulpal chamber was sampled every 30 min for 5 h and at 24 h using the limulus lysate assay. In four specimens the initial appearance of endotoxin in the effluent ranged from 15 min to 4 1/2 h. In two specimens the concentration of endotoxin in the effluent leveled off in 4 1/2 and 5 h, respectively, whereas in another two the concentration continued to increase throughout the experiment. In one specimen no endotoxin was detected. The results indicate that endotoxin is capable of passing through 0.5 mm of dentin.

Macromolecular leakage beneath full cast crowns. Part III: The diffusion of lipopolysaccharide and dextran.

STATEMENT OF PROBLEM: Most microleakage studies have used low molecular weight dyes or isotopes rather than clinically relevant materials, such as lipopolysaccharides or cell wall materials, that have been shown to provoke inflammatory reactions in the dental pulp. PURPOSE: This study evaluated the leakage (diffusion) of lipopolysaccharide and dextran beneath cast gold crowns luted with 3 cements. MATERIALS AND METHODS: Thirty extracted molars were prepared for crowns. Ten crowns with access ports (facial or lingual) were cast in gold and luted with zinc phosphate, glass ionomer, and an adhesive resin cement onto their preparations. Teeth and crowns with filters inserted into the ports were immersed in a solution of labeled macromolecules (TRITC-LPS, FITC-dextran) and evaluated for leakage at 2 weeks, and 1, 2, 3, 4, 5, and 6 months. Filters were retrieved and analyzed with fluorescent microscopy. RESULTS: All filters retrieved from crowns luted with zinc phosphate, glass ionomer, and adhesive resin cements demonstrated no detectable leakage and were negative for both FITC-dextran and TRITC-lipopolysaccharide at all evaluation periods. CONCLUSION: Leakage of lipopolysaccharides and dextran did not occur during the period of this study. For the length of this investigation, zinc phosphate, Ketac-Cem, and C&B-Metabond were equally effective at preventing leakage of detectable molecular concentrations of lipopolysaccharide and dextran to the level of the access ports.

Induction of IL-1 during hemodialysis: transmembrane passage of intact endotoxins (LPS).

Circulating monocytes of patients undergoing chronic hemodialysis are triggered to produce interleukin-1 (IL-1) in vivo. Intradialytic induction of IL-1 is associated with complement activation in patients dialyzed with first-use cellulose membranes. Chronic stimulation of IL-1 production occurs because of an yet unidentified mechanism in patients dialyzed with high permeability membranes. The present study demonstrates that intact bacterial lipopolysaccharide (LPS) molecules may cross cuprophan, AN69 and polysulfone membranes under in vitro conditions simulating in vivo hemodialysis. The experiments used purified LPS from Neisseria meningitidis and LPS from Pseudomonas testosteroni, a bacterial strain grown out from a clinically used dialysate. LPS were purified to homogeneity and radiolabeled. Transmembrane passage of 3H-labeled LPS was observed within the first five minutes of dialysis. A total of 0.1 to 1% of 3H-labeled LPS were recovered in the dialysate compartment after one hour of dialysis. High amounts of LPS, representing 40 to 70% of the amount originally present in the dialysate, were absorbed onto high permeability membranes. Low amounts of LPS were absorbed onto cuprophan membranes. The amount of LPS absorbed decreased with the concentration of LPS in the dialysate. LPS recovered from the blood compartment exhibited the same molecular weight as that used to contaminate the dialysate. Biochemically detectable transmembrane passage of LPS was not associated with that of material detectable using the limulus amebocyte lysate (LAL) assay. An IL-1-inducing activity was, however, detected in the blood compartment upon dialysis with high permeability membranes, as previously found by others with cuprophan membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Macromolecular leakage benath full cast crowns. Part II: The diffusion of lipopolysaccharide and dextran.

Fifteen extracted molars were prepared for crowns. Crowns with access ports (one facial, one lingual) were cast in gold. Teeth and crowns luted with provisional cement with filters inserted into the ports were immersed in a solution of labeled macromolecules (FITC-dextran, TRITC-LPS) and evaluated for leakage. Filters were retrieved and analyzed by use of fluorescent microscopy. Leakage of LPS and dextran occurred as early as 2 weeks beneath crowns luted with a provisional cement (NoGenol).

The effect of Triton X-100 on the in-vitro susceptibility of methicillin-resistant Staphylococcus aureus to oxacillin.

The effect of the non-ionic detergent, Triton X-100, on the in-vitro activity of oxacillin against methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) strains of Staphylococcus aureus was investigated. In the presence of Triton X-100, the MICs of oxacillin for both MRSA and MSSA isolates were reduced; this enhancing effect was particularly marked for the MRSA strains. Triton X-100 therefore counteracted the resistance to methicillin encoded by mecA. In the presence of oxacillin at subinhibitory concentrations, Triton X-100 induced the bacteriolysis of MRSA and potentiated the autolysis of these organisms. However, the detergent had no effect on the bacteriolytic enzyme profile or the susceptibility of the bacterial cell wall to bacteriolytic enzymes, nor did it promote the binding of oxacillin to the penicillin-binding protein (PBP) 2A. On the other hand, it stimulated the release from the bacteria of acylated lipoteichoic acid (LTA), a putative endogenous regulator of autolysins. Autolytic enzyme-deficient MRSA mutants were equally as sensitive as the parent strain to the effect of Triton X-100 on susceptibility to oxacillin. These results indicate that the enhanced in-vitro activity of oxacillin against MRSA in the presence of Triton X-100 cannot be accounted for simply by the induction of bacteriolysis following activation of autolytic enzymes by the detergent-stimulated release of LTA.

Cytotoxicity and sealing properties of four classes of endodontic sealers evaluated by succinic dehydrogenase activity and confocal laser scanning microscopy.

The objectives of this study were to evaluate the cytotoxicity and sealing properties of four classes of endodontic sealers (PCS/Kerr, RoekoSeal/Roeko, TopSeal/Dentsply, and EndoREZ/Ultradent). For cytotoxicity testing (MTT method), the materials were either placed immediately in contact with cultured cells or 24 h after setting, then evaluated at three subsequent time points (24 h, 48 h, or 1 wk). For the leakage study, extracted human roots were obturated with acrylic cones and sealers and immersed for 48 h into rhodamine-labeled lipopolysaccharide. The roots were then observed under a confocal laser scanning microscope to estimate (semiquantitatively) the presence of the rhodamine-lipopolysaccharide (LPS) inside the canal. The results showed that cytotoxicity generally increased with time, and that most materials pose significant cytotoxic risks, particularly in the freshly mixed condition. Further, all materials showed significant leakage although there was large variation among teeth. Overall, the silicon-based material (Roeko Seal) was less cytotoxic and more effective in sealing root canals against LPS leakage than other materials.