Liposomal Hepatitis A Vaccine and Liposomal Multiantigen Combination Vaccines

Abstract

Many antigens are only weak immunogens, and they require adjuvants to reach a satisfactory immune response as vaccines. This is especially true for modern antigens, such as products of gene technology or synthetic peptides. Also, classically produced antigens such as diphtheria or tetanus-antigens are coupled on aluminium phosphate or aluminium hydroxide – to this day practically the only adjuvant class which is routinely used in vaccinology. Aluminium phosphate was first used in the 1920s as an immunopotentiator for the diphtheria vaccine. Since then, and up until now, it has not been possible to fully replace this unsatisfactory adjuvant. exclusion of epitope-specific suppression by the individual components, it was evident that particularly the diphtheria and tetanus constituents exerted “antigenic competition” on the HAV antigen and possibly also on the HBs antigen. By reducing the diphtheria and tetanus toxoid subunit molecules per IRIV particle, it was possible not only to increase significantly the immune response to these antigens but also to remove completely the antigenic competition on the HAV and HBs antigens. Once the optimum composition of the vaccine had been achieved (careful dosing of antigens per IRIV particle), an immunological effect clearly superior to that of comparable, aluminium-adsorbed products was obtained (Table II). The tolerability of this “supercombi vaccine” was significantly better than the commercial alum adsorbed products (Table III).     

Macrophage Activation by Muramyl Dipeptide as Measured by Macrophage Spreading and Attachment

A synthetic bacterial cell wall constituent, muramyl dipeptide (MDP), was found to induce the enhancement of macrophage spreading and attachment on glass or plastic surfaces. Macrophages exposed to bacterial lipopolysaccharide or lymphokine-containing cell supernatants showed similar enhancement. This finding supports the view that MDP activates macrophages. MDP was also found to enhance the viability of macrophages but to inhibit ³H-thymidine incorporation by macrophages.     

B Cell Infection and Activation by Rabies Virus-Based Vaccines

Replication-deficient rabies viruses (RABV) are promising rabies postexposure vaccines due to their prompt and potent stimulation of protective virus neutralizing antibody titers, which are produced in mice by both T-dependent and T-independent mechanisms. To promote such early and robust B cell stimulation, we hypothesized that live RABV-based vaccines directly infect B cells, thereby activating a large pool of antigen-presenting cells (APCs) capable of providing early priming and costimulation to CD4⁺ T cells. In this report, we show that live RABV-based vaccine vectors efficiently infect naive primary murine and human B cells ex vivo. Infection of B cells resulted in the significant upregulation of early markers of B cell activation and antigen presentation, including CD69, major histocompatibility complex class II (MHC-II), and CD40 in murine B cells or HLA-DR and CD40 in human B cells compared to mock-infected cells or cells treated with an inactivated RABV-based vaccine. Furthermore, primary B cells infected with a live RABV expressing ovalbumin were able to prime and stimulate naive CD4⁺ OT-II T cells to proliferate and to secrete interleukin-2 (IL-2), demonstrating a functional consequence of B cell infection and activation by live RABV-based vaccine vectors. We propose that this direct B cell stimulation by live RABV-based vaccines is a potential mechanism underlying their induction of early protective T cell-dependent B cell responses, and that designing live RABV-based vaccines to infect and activate B cells represents a promising strategy to develop a single-dose postexposure rabies vaccine where the generation of early protective antibody titers is critical.     

Myelin-Derived Lipids Modulate Macrophage Activity by Liver X Receptor Activation

Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system in which macrophages and microglia play a central role. Foamy macrophages and microglia, containing degenerated myelin, are abundantly found in active multiple sclerosis lesions. Recent studies have described an altered macrophage phenotype after myelin internalization. However, it is unclear by which mechanisms myelin affects the phenotype of macrophages and how this phenotype can influence lesion progression. Here we demonstrate, by using genome wide gene expression analysis, that myelin-phagocytosing macrophages have an enhanced expression of genes involved in migration, phagocytosis and inflammation. Interestingly, myelin internalization also induced the expression of genes involved in liver-X-receptor signaling and cholesterol efflux. In vitro validation shows that myelin-phagocytosing macrophages indeed have an increased capacity to dispose intracellular cholesterol. In addition, myelin suppresses the secretion of the pro-inflammatory mediator IL-6 by macrophages, which was mediated by activation of liver-X-receptor β. Our data show that myelin modulates the phenotype of macrophages by nuclear receptor activation, which may subsequently affect lesion progression in demyelinating diseases such as multiple sclerosis.     

TMAO通过CD147/MMPs通路诱导巨噬细胞活化

肠道菌群代谢产物氧化三甲胺(trimethylamine N-oxide,TMAO)可通过多种途径促进动脉粥样硬化(atherosclerosis,AS)的进展,现研究发现,在临床上其与斑块稳定性存在密切联系,但其分子机制目前尚不明确.金属蛋白酶诱导因子(extracellular matrix metalloprot...     

Iron-Melanin Interaction and Lipid Peroxidation: Implications for Parkinson''s Disease

The vulnerability of substantia nigral (SN) melaninized dopamine neurons to neurodegeneration in Parkinson's disease and the selective increases of iron and basal lipid peroxidation in SN indicate that iron-melanin interaction could be crucial to the pathogenesis of this disease. The present study describes, for the first time, the identification and characterization of a high-affinity (KD = 13 nM) and a lower affinity (KD = 200 nM) binding site for iron on dopamine melanin. The binding of iron to melanin is dependent on pH and the concentration of melanin. Iron chelators, U74500A, desferrioxamine, and to less extent 1,10-phenanthroline and chlorpromazine, but not the Parkinson-inducing neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, can inhibit the binding of iron to melanin and iron-induced lipid peroxidation. Although melanin alone diminishes basal lipid peroxidation in rat cortical homogenates, it can also potentiate that initiated by iron, a reaction inhibited by desferrioxamine. In the absence of an identifiable exogenous or endogenous neurotoxin in idiopathic Parkinson's disease, iron-melanin interaction in pars compacta of SN may be a strong candidate for the cytotoxic component of oxygen radical-induced neurodegeneration of melaninized dopamine neurons.     

Leishmania donovani Infection Enhances Lateral Mobility of Macrophage Membrane Protein Which Is Reversed by Liposomal Cholesterol

BackgroundThe protozoan parasite Leishmania donovani (LD) reduces cellular cholesterol of the host possibly for its own benefit. Cholesterol is mostly present in the specialized compartment of the plasma membrane. The relation between mobility of membrane proteins and cholesterol depletion from membrane continues to be an important issue. The notion that leishmania infection alters the mobility of membrane proteins stems from our previous study where we showed that the distance between subunits of IFNγ receptor (R1 and R2) on the cell surface of LD infected cell is increased, but is restored to normal by liposomal cholesterol treatment.Conclusions/SignificancesTo our knowledge this is the first direct demonstration that LD parasites during their intracellular life cycle increases lateral mobility of membrane proteins and decreases F-actin level in infected macrophages. Such defects may contribute to ineffective intracellular signaling and other cellular functions.     

Macrophage Activation by Muramyl Dipeptide (MDP) without Lymphocyte Participation

An increase in the numbers of spread macrophages caused by macrophage stimulants was found to be a very sensitive measure for macrophage activation. Muramyl dipeptide (MDP), bacterial lipopolysaccharide (LPS), and lymphokines were found to activate macrophages dose-dependently as measured by this parameter. Macrophage activation by MDP was strictly dependent on its adjuvant-active stereochemically specific structures. Macrophage activation by MDP and LPS occurred without lymphocyte participation. It is suggested that LPS also activates macrophages via lymphocytes.     

Early Activation of MAP Kinases by Influenza A Virus X-31 in Murine Macrophage Cell Lines

Early molecular responses to Influenza A (FLUA) virus strain A/X-31 H3N2 in macrophages were explored using J774.A1 and RAW 264.7 murine cell lines. NF-kappa B (NFκB) was reported to be central to FLUA host-response in other cell types. Our data showed that FLUA activation of the classical NFκB dependent pathway in these macrophages was minimal. Regulator proteins, IkappaB-alpha and –beta (IκBα, IκBβ), showed limited degradation peaking at 2 h post FLUA exposure and p65 was not observed to translocate from the cytoplasm to the nucleus. Additionally, the non-canonical NFκB pathway was not activated in response to FLUA. The cells did display early increases in TNFα and other inflammatory cytokine and chemokine production. Mitogen activated phosphokinase (MAPK) signaling pathways are also reported to control production of inflammatory cytokines in response to FLUA. The activation of the MAPKs, cJun kinases 1 and 2 (JNK 1/2), extracellular regulated kinases 1 and 2 (ERK 1/2), and p38 were investigated in both cell lines between 0.25 and 3 h post-infection. Each of these kinases showed increased phosphorylation post FLUA exposure. JNK phosphorylation occurred early while p38 phosphorylation appeared later. Phosphorylation of ERK 1/2 occurred earlier in J774.A1 cells compared to RAW 264.7 cells. Inhibition of MAPK activation resulted in decreased production of most FLUA responsive cytokines and chemokines in these cells. The results suggest that in these monocytic cells the MAPK pathways are important in the early response to FLUA.     

Macrophage Activation by Tetrahymena pyriformis II. Active Protein Fractions from Tetrahymena

ABSTRACT. Water-soluble and 0.6 M KCl-soluble protein fractions prepared from Tetrahymena pyriformis , when inoculated into mice, could effectively induce activated macrophages having the ability to kill Toxoplasma gondii in vitro. This effect was not induced by other proteins tested, such as bovine serum albumin, pepsin from porcine stomach mucosa and chicken egg-white lysozyme, nor by muramyl dipeptide (MDP), a potent immunoadjuvant. Five fractions obtained by DEAE-Sephadex chromatography of the water-soluble protein fraction were compared with regard to induction of toxoplasmacidal activity in macrophages. The first peak was most effective for activation of macrophages. Five fractions obtained by chromatography of the 0.6 M KCl-soluble protein fraction were also examined and it was found that the first peak had the activity. No marked difference in activity was observed between the active fractions of water-soluble and 0.6 M KCl-soluble protein fractions. For practical use, we focused on the water-soluble active fraction. The minimum effective dose of the active fraction was 100 μg and the fraction could activate macrophages directly in vitro. Four fractions obtained by gel filtration of the active fraction on Sephadex G-200 were compared and the first peak had the activity. The first peak contained a single protein, revealed by SDS-polyacrylamide gel electrophoresis; its apparent molecular weight was 64,000.     

Triglyceride Blisters in Lipid Bilayers: Implications for Lipid Droplet Biogenesis and the Mobile Lipid Signal in Cancer Cell Membranes

Triglycerides have a limited solubility, around 3%, in phosphatidylcholine lipid bilayers. Using millisecond-scale course grained molecular dynamics simulations, we show that the model lipid bilayer can accommodate a higher concentration of triolein (TO) than earlier anticipated, by sequestering triolein molecules to the bilayer center in the form of a disordered, isotropic, mobile neutral lipid aggregate, at least 17 nm in diameter, which forms spontaneously, and remains stable on at least the microsecond time scale. The results give credence to the hotly debated existence of mobile neutral lipid aggregates of unknown function present in malignant cells, and to the early biogenesis of lipid droplets accommodated between the two leaflets of the endoplasmic reticulum membrane. The TO aggregates give the bilayer a blister-like appearance, and will hinder the formation of multi-lamellar phases in model, and possibly living membranes. The blisters will result in anomalous membrane probe partitioning, which should be accounted for in the interpretation of probe-related measurements.     

Systemic and Mucosal T-Lymphocyte Activation Induced by Recombinant Adenovirus Vaccines in Rhesus Monkeys

The administration of vectors designed to elicited cell-mediated immune responses may have other consequences that are clinically significant. To explore this possibility, we evaluated T-cell activation during the first 2 months after recombinant adenovirus serotype 5 (rAd5) prime or boost immunizations in rhesus monkeys. We also evaluated the kinetics of T-lymphocyte activation in both the systemic and the mucosal compartments after rAd5 administration in monkeys with preexisting immunity to Ad5. The rAd5 immunization induced lower-frequency Gag epitope-specific CD8⁺ T cells in the colonic mucosa than in the peripheral blood. There was evidence of an expansion of the simian immunodeficiency virus Gag-specific CD8⁺ T-cell responses, but not the Ad5 hexon-specific T-cell responses, following a homologous rAd5 boost. A striking but transient T-lymphocyte activation in both the systemic and the mucosal compartments of rhesus monkeys was observed after rAd5 immunization. These findings indicate that the administration of a vaccine vector such as Ad5 can induce a global activation of T cells.Considerable effort has been invested in the development of vaccine strategies for eliciting cell-mediated immune responses to human immunodeficiency virus (HIV). Studies in simian immunodeficiency virus (SIV)/SHIV-infected nonhuman primates and HIV-infected humans demonstrated a central role for cell-mediated immune responses in the containment of HIV replication (1, 12). These findings led to the hypothesis that vaccine-elicited cell-mediated immunity might contribute to improved control of HIV in infected individuals. Studies in the SIV and SHIV/macaque models have supported this hypothesis, demonstrating a decrease in peak plasma virus RNA levels during primary infection, protection against memory CD4⁺ T-cell lymphocyte loss, and prolonged survival of monkeys that had vaccine-elicited cell-mediated immunity to the virus prior to challenge (8, 15, 16).Despite promising results in preclinical nonhuman primate studies, a prophylactic HIV vaccine trial of the Merck recombinant adenovirus serotype 5 (rAd5) vector expressing HIV gag, pol, and nef genes (STEP trial) was recently halted due to a 2.3-fold increase of HIV acquisition in vaccinees with preexisting neutralizing antibodies (NAbs) to Ad5 (2, 9, 10). This finding raised the possibility that T lymphocytes that are activated in response to vaccination might represent an increased pool of potential targets for HIV infection, and the persistence of such activated cells may increase the susceptibility of the vaccinated individual to acquiring an HIV infection (5, 11). HIV replicates most readily in activated, CCR5⁺CD4⁺ T lymphocytes. It has been suggested that vaccines that elicit potent cellular immune responses may also activate subpopulations of CD4⁺ T lymphocytes. In fact, in the aftermath of the failed STEP trial, it was proposed that the activation of Ad5-specific T cells in individuals with prior Ad5 immunity may have contributed to their increased acquisition of HIV after vaccination.The contribution of cellular activation in mucosal tissues to acquisition of HIV remains unexplored (2). HIV transmission occurs most often across mucosal barriers. There is increasing evidence that CD4⁺ T lymphocytes are among the first cells infected during the transmission event (4). Activation of mucosal populations of lymphocytes as a consequence of vaccination could contribute to increasing the incidence of HIV transmission at a mucosal site.To examine these issues, the present study was initiated to explore vaccine-induced activation of T-lymphocyte populations in rhesus monkeys. The character and kinetics of the activation of both circulating and mucosal T-lymphocyte populations were evaluated after immunization with a variety of immunogens. These experiments demonstrate a striking but transient T-lymphocyte activation induced by adenovirus-based vaccine vectors in both the systemic and mucosal compartments of rhesus monkeys.     

Liposomal nanocarriers for statins: A pharmacokinetic and pharmacodynamics appraisal

Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, are a well-known class of drug with beneficial therapeutic effects in cardiovascular disease and lipid disorders and have potential use against cancer. However, the bioavailability of statins is hampered due to low aqueous solubility and rapid metabolism. To improve pharmacokinetic profiles of statins, development of drug delivery systems is promising. Hence, the use of liposomes for selective delivery of statins to a selected site or for bioavailability enhancement is an effective strategy to increase statin therapeutic effects. Moreover, liposomal delivery can reduce the required dose of statins especially in terms of antitumor effects. Liposomes, because of their unique properties and biphasic and amphiphilic nature, have attracted much interest and can be considered as a suitable choice for delivery of both hydrophilic and lipophilic statins. In this review article, we focus on liposomes and evaluate the effects of different liposomal delivery systems, based on differences in size, phospholipid composition, circulation half-life, and cholesterol content, on statin function.     

Macrophage Activation by Tetrahymena pyriformis. I. Active Subcellular Fractions of Tetrahymena

Experiments were carried out to determine what subcellular fractions of Tetrahymena pyriformis could, after inoculation into mice, activate macrophages to kill Toxoplasma gondii in vitro. Peritoneal macrophages from mice inoculated intraperitoneally with cilia, pellicles, mitochondria, and microsomes exhibited strong toxoplasmacidal activity and had an enhanced capacity to release hydrogen peroxide (H₂O₂) by stimulation of a membrane-active agent as compared with resident macrophages. In contrast, macrophages from mice inoculated with macronuclei and postmicrosomal supernatant showed no toxoplasmacidal activity and a low level of H₂O₂ release. Similar dose response was observed on the active subcellular fractions with regard to the degree of macrophage activation. Treatment of the active subcellular fractions with heating and trypsin markedly reduced their activity.     

CR3 and Dectin-1 Collaborate in Macrophage Cytokine Response through Association on Lipid Rafts and Activation of Syk-JNK-AP-1 Pathway

Collaboration between heterogeneous pattern recognition receptors (PRRs) leading to synergistic coordination of immune response is important for the host to fight against invading pathogens. Although complement receptor 3 (CR3) and Dectin-1 are major PRRs to detect fungi, crosstalk between these two receptors in antifungal immunity is largely undefined. Here we took advantage of Histoplasma capsulatum which is known to interact with both CR3 and Dectin-1 and specific particulate ligands to study the collaboration of CR3 and Dectin-1 in macrophage cytokine response. By employing Micro-Western Array (MWA), genetic approach, and pharmacological inhibitors, we demonstrated that CR3 and Dectin-1 act collaboratively to trigger macrophage TNF and IL-6 response through signaling integration at Syk kinase, allowing subsequent enhanced activation of Syk-JNK-AP-1 pathway. Upon engagement, CR3 and Dectin-1 colocalize and form clusters on lipid raft microdomains which serve as a platform facilitating their cooperation in signaling activation and cytokine production. Furthermore, in vivo studies showed that CR3 and Dectin-1 cooperatively participate in host defense against disseminated histoplasmosis and instruct adaptive immune response. Taken together, our findings define the mechanism of receptor crosstalk between CR3 and Dectin-1 and demonstrate the importance of their collaboration in host defense against fungal infection.     

Cleavage of Type I Collagen by Fibroblast Activation Protein-α Enhances Class A Scavenger Receptor Mediated Macrophage Adhesion

Pathophysiological conditions such as fibrosis, inflammation, and tumor progression are associated with modification of the extracellular matrix (ECM). These modifications create ligands that differentially interact with cells to promote responses that drive pathological processes. Within the tumor stroma, fibroblasts are activated and increase the expression of type I collagen. In addition, activated fibroblasts specifically express fibroblast activation protein-α (FAP), a post-prolyl peptidase. Although FAP reportedly cleaves type I collagen and contributes to tumor progression, the specific pathophysiologic role of FAP is not clear. In this study, the possibility that FAP-mediated cleavage of type I collagen modulates macrophage interaction with collagen was examined using macrophage adhesion assays. Our results demonstrate that FAP selectively cleaves type I collagen resulting in increased macrophage adhesion. Increased macrophage adhesion to FAP-cleaved collagen was not affected by inhibiting integrin-mediated interactions, but was abolished in macrophages lacking the class A scavenger receptor (SR-A/CD204). Further, SR-A expressing macrophages localize with activated fibroblasts in breast tumors of MMTV-PyMT mice. Together, these results demonstrate that FAP-cleaved collagen is a substrate for SR-A-dependent macrophage adhesion, and suggest that by modifying the ECM, FAP plays a novel role in mediating communication between activated fibroblasts and macrophages.