lipid-a and Disease-Models--Animal

Airway inflammatory disorders are prevalent diseases in need of better management and new therapeutics. Immunotherapies offer a solution to the problem of corticosteroid resistance. Areas covered: The current review focuses on lipopolysaccharide (Gram-negative bacterial endotoxin)-mediated inflammation in the lung and the animal models used to study related diseases. Endotoxin-induced lung pathology is usually initiated by antigen presenting cells (APC). We will discuss different subsets of APC including lung dendritic cells and macrophages, and their role in responding to endotoxin and environmental challenges. Expert commentary: The pharmacotherapeutic considerations to combat airway inflammation should cost-effectively improve quality of life with sustainable and safe strategies. Selectively targeting APCs in the lung offer the potential for a promising new strategy for the better management and treatment of inflammatory lung disease.

Topics: Allergens; Animals; Anti-Inflammatory Agents; Asthma; Bacteria; Cell Adhesion Molecules; Disease Models, Animal; Drug Carriers; Endotoxins; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Immunity, Innate; Lipid A; Lipopolysaccharides; Myeloid Cells; Nanoparticles; Neutrophils; Protease Inhibitors; Pulmonary Disease, Chronic Obstructive; Respiratory Distress Syndrome; Toll-Like Receptors

Adjuvants are used to improve vaccine immunogenicity and efficacy by enhancing antigen presentation to antigen-specific immune cells with the aim to confer long-term protection against targeted pathogens. Adjuvants have been used in vaccines for more than 90 years. Combinations of immunostimulatory molecules, such as in the Adjuvant System AS01, have opened the way to the development of new or improved vaccines. Areas covered: AS01 is a liposome-based vaccine adjuvant system containing two immunostimulants: 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and the saponin QS-21. Here we describe studies investigating the mode of action of AS01, and consider the role of AS01 in enhancing specific immune responses to the antigen for selected candidate vaccines targeting malaria and herpes zoster. The effects of AS01 are rapid and transient, being localized to the injected muscle and draining lymph node. AS01 is efficient at promoting CD4

Topics: Adjuvants, Immunologic; Animals; CD4-Positive T-Lymphocytes; Disease Models, Animal; Drug Combinations; Herpes Zoster Vaccine; Humans; Lipid A; Liposomes; Malaria Vaccines; Saponins

Bacterial lipopolysaccharide (LPS) and its active component, lipid A, have been used either alone or as adjuvant in therapeutic anticancer vaccines. Lipid A induces various transcription factors via intracellular signaling cascades initiated by their receptor CD14-TLR4. These events lead to the synthesis of cytokines, which either have direct cytotoxic effect or stimulate the immune system. Their antitumoral effect has been demonstrated in animal models as well as clinical trials. Studies in animal models showed that their antitumoral effect relies mostly on the generation of an effective immune response. In humans, the antitumoral effect was correlated with an antibody response and cell-mediated cytotoxicity. So far, some encouraging results have been achieved in phase I and II clinical trials with regards to response and stabilization of the disease, but an expansion of the studies and trials is needed to find the best conditions for their clinical application.

Topics: Animals; Antineoplastic Agents; Disease Models, Animal; Humans; Lipid A; Neoplasms, Experimental

Topics: Adjuvants, Immunologic; Animals; Antineoplastic Agents; Clinical Trials as Topic; Disease Models, Animal; Humans; Lipid A; Neoplasms; Neoplasms, Experimental; Vaccination

Despite considerable efforts in the past quarter century to improve therapy for sepsis, mortality rates remain unacceptably high. Microbe-derived constituents can induce the host to produce many mediators that can contribute to immune dysregulation, tissue damage, and death. Although endotoxin-mediated events are clearly important in gram-negative infections, gram-positive bacteria can also play a dominant role. Understanding the interplay of microbial constituents and host immune or inflammatory responses prompted a meeting at Rockefeller University in May 1998. Participants discussed the relative merits of a "2-hit" hypothesis to explain the course of lethal septic shock and a "multihit" synergistic threshold hypothesis. Recommendations include the following: (1) developing animal models that closely mimic human sepsis; (2) further investigating antibiotic effects on bacteria; (3) assessing the relationships between endotoxin, prokaryotic DNA, and peptidoglycan (i.e., independent, additive, or synergistic) in inducing host responses; and (4) developing new strategies to improve outcomes. Studies are needed to better define which and how different microbial constituents lead to sepsis and to provide critical leads for therapeutic intervention.

Topics: Amino Acid Sequence; Animals; Carbohydrate Sequence; Disease Models, Animal; DNA, Bacterial; Endotoxins; Gram-Positive Bacteria; Humans; Inflammation Mediators; Lipid A; Models, Biological; Peptidoglycan; Sepsis

Monophosphoryl lipid A represents a novel agent capable of enhancing myocardial tolerance to ischemia/reperfusion injury. This cardioprotective activity of MLA manifests itself as a reduction in infarct size, myocardial stunning and dysrhythmias in multiple animal species. The drug appears to be efficacious in dogs and rabbits at doses of 10-35 micrograms/kg, with larger doses seemingly required in the rat. In the rabbit infarct model, protection appears 6 h following drug administration and lasts for 36 h. Although multifactorial mechanisms of ischemic tolerance may be induced by MLA, current evidence suggests that MLA's cardioprotective effects involve myocardial iNOS enzyme activation with nitric oxide coupled activation of myocardial KATP channels upon ischemic challenge. Monophosphoryl lipid A is presently being evaluated in Phase 2 clinical trials in patients undergoing cardiopulmonary bypass associated with coronary artery bypass engraftment or aortic valve replacement or reconstruction. Severity of lethal and reversible myocardial injury and dysrhythmia are study endpoints. Although further clinical testing will establish the utility of MLA as a cardioprotectant against ischemia/reperfusion injury in the human, presently this agent is proving very useful in expanding our understanding of mechanisms responsible for delayed cardiac preconditioning against ischemia/reperfusion injury.

Topics: Animals; Clinical Trials, Phase II as Topic; Disease Models, Animal; Humans; Ischemic Preconditioning, Myocardial; Lipid A; Myocardial Reperfusion Injury; Myocardium

The unknowns persisting in the understanding of the mode of action of anti-core lipopolysaccharide antibodies are discussed, and a study of two anti-lipid A monoclonal antibodies is reviewed. This article also critically analyzes the results of the recent clinical trials with monoclonal antibodies.

Topics: Animals; Antibodies, Bacterial; Antibodies, Monoclonal; Disease Models, Animal; Epitopes; Escherichia coli; Gram-Negative Bacterial Infections; Humans; Immunoglobulins, Intravenous; Immunotherapy; Lipid A; Lipopolysaccharides

Topics: Acute Kidney Injury; Animals; Anti-Bacterial Agents; Disease Models, Animal; Endotoxins; Glomerular Filtration Rate; Glycerol; Humans; Kidney; Lipid A; Renal Circulation; Shock, Septic; Vasoconstriction

Treatment of visceral leishmaniasis (VL) is compromised by drug toxicity, high cost and/or the emergence of resistant strains. Though canine vaccines are available, there are no licensed prophylactic human vaccines. One strategy to improve clinical outcome for infected patients is immunotherapy, which associates a chemotherapy that acts directly to reduce parasitism and the administration of an immunogen-adjuvant that activates the host protective Th1-type immune response. In this study, we evaluated an immunotherapy protocol in a murine model by combining recombinant (r)LiHyp1 (a hypothetical amastigote-specific Leishmania protein protective against Leishmania infantum infection), with monophosphoryl-lipid A (MPLA) as adjuvant and amphotericin B (AmpB) as reference antileishmanial drug. We used this protocol to treat L. infantum infected-BALB/c mice, and parasitological, immunological and toxicological evaluations were performed at 1 and 30 days after treatment. Results showed that mice treated with rLiHyp1/MPLA/AmpB presented the lowest parasite burden in all organs evaluated, when both a limiting dilution technique and qPCR were used. In addition, these animals produced higher levels of IFN-γ and IL-12 cytokines and IgG2a isotype antibody, which were associated with lower production of IL-4 and IL-10 and IgG1 isotype. Furthermore, low levels of renal and hepatic damage markers were found in animals treated with rLiHyp1/MPLA/AmpB possibly reflecting the lower parasite load, as compared to the other groups. We conclude that the rLiHyp1/MPLA/AmpB combination could be considered in future studies as an immunotherapy protocol to treat against VL.

Topics: Adjuvants, Immunologic; Amebicides; Amphotericin B; Animals; Clinical Protocols; Disease Models, Animal; Drug Therapy, Combination; Female; Immunotherapy; Leishmaniasis, Visceral; Lipid A; Mice; Mice, Inbred BALB C; Protozoan Proteins; Recombinant Proteins

Topics: Animals; Apolipoproteins E; Diet, High-Fat; Disease Models, Animal; Down-Regulation; Food Additives; Genistein; Humans; Intracellular Signaling Peptides and Proteins; Lipid A; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Plaque, Atherosclerotic; RAW 264.7 Cells; Signal Transduction

Osteosarcoma (OS) is the most common malignant bone tumor and the prognosis of advanced cases is still poor. Recently, there have been several reports suggesting the relationship between innate immunity and OS, but the detailed mechanism is unknown. We demonstrate the relationship between OS and Toll-like receptor 4 (TLR4) which is one of the most important factors in innate immunity.. We established a syngenic mouse tumor model using C3H/HeN, C3H/HeJ mouse and a highly metastatic OS cell line, LM8. TLR4 activation with lipopolysaccharide (LPS) was performed on both mice and its influence on the progression of OS was evaluated. We also performed CD8 + cells depletion to examine the influence on TLR4 activation effects.. Tumor volume of C3H/HeN mice was significantly smaller and overall survival of C3H/HeN mice was significantly longer than C3H/HeJ mice. We found more CD8+ cells infiltrating in lung metastases of C3H/HeN mice and depletion of CD8+ cells canceled the antitumor effects of LPS.. TLR4 activation by LPS increased CD8+ cells infiltrating into lung metastases and suppressed OS progression in the mouse model. TLR4 activation may suppress the progression of OS via stimulating CD8+ cells and can be expected as a novel treatment for OS.

Topics: Adjuvants, Immunologic; Adolescent; Animals; Antineoplastic Combined Chemotherapy Protocols; Bone Neoplasms; Cell Line, Tumor; Chemotherapy, Adjuvant; Child; Child, Preschool; Disease Models, Animal; Disease Progression; Female; Humans; Kaplan-Meier Estimate; Lipid A; Lipopolysaccharides; Lung Neoplasms; Lymphocyte Depletion; Male; Mice; Middle Aged; Osteosarcoma; Prognosis; Progression-Free Survival; Signal Transduction; T-Lymphocytes, Cytotoxic; Toll-Like Receptor 4; Tumor Burden; Young Adult

Radiation protection on male testis is an important task for ionizing radiation-related workers or people who receive radiotherapy for tumours near the testicle. In recent years, Toll-like receptors (TLRs), especially TLR4, have been widely studied as a radiation protection target. In this study, we detected that a low-toxicity TLR4 agonist monophosphoryl lipid A (MPLA) produced obvious radiation protection effects on mice testis. We found that MPLA effectively alleviated testis structure damage and cell apoptosis induced by ionizing radiation (IR). However, as the expression abundance differs a lot in distinct cells and tissues, MPLA seemed not to directly activate TLR4 singling pathway in mice testis. Here, we demonstrated a brand new mechanism for MPLA producing radiation protection effects on testis. We observed a significant activation of TLR4 pathway in macrophages after MPLA stimulation and identified significant changes in macrophage-derived exosomes protein expression. We proved that after MPLA treatment, macrophage-derived exosomes played an important role in testis radiation protection, and specially, G-CSF and MIP-2 in exosomes are the core molecules in this protection effect.

Topics: Abnormalities, Radiation-Induced; Animals; Disease Models, Animal; Exosomes; Humans; Lipid A; Male; Mice; Radiation Protection; Testis; Toll-Like Receptor 4

Following an episode of cholera, a rapidly dehydrating, watery diarrhea caused by the Gram-negative bacterium, Vibrio cholerae O1, humans mount a robust anti-lipopolysaccharide (LPS) antibody response that is associated with immunity to subsequent re-infection. In neonatal mouse and rabbit models of cholera, passively administered anti-LPS polyclonal and monoclonal (MAb) antibodies reduce V. cholerae colonization of the intestinal epithelia by inhibiting bacterial motility and promoting vibrio agglutination. Here we demonstrate that human anti-LPS IgG MAbs also arrest V. cholerae motility and induce bacterial paralysis. A subset of those MAbs also triggered V. cholerae to secrete an extracellular matrix (ECM). To identify changes in gene expression that accompany antibody exposure and that may account for motility arrest and ECM production, we subjected V. cholerae O1 El Tor to RNA-seq analysis after treatment with ZAC-3 IgG, a high affinity MAb directed against the core/lipid A region of LPS. We identified > 160 genes whose expression was altered following ZAC-3 IgG treatment, although canonical outer membrane stress regulons were not among them. ompS (VCA1028), a porin associated with virulence and indirectly regulated by ToxT, and norR (VCA0182), a σ54-dependent transcription factor involved in late stages of infection, were two upregulated genes worth noting.

Topics: Agglutination; Animals; Antibodies, Bacterial; Antibodies, Monoclonal; Bacterial Outer Membrane Proteins; Bacterial Proteins; Cholera; Cholera Toxin; Disease Models, Animal; Gene Deletion; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genes, Bacterial; Host-Pathogen Interactions; Humans; Immunoglobulin G; Lipid A; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Rabbits; Transcription Factors; Transcriptome; Vibrio cholerae O1; Virulence

Topics: Adjuvants, Immunologic; Animals; CD4-Positive T-Lymphocytes; Cytokines; Disease Models, Animal; Drug Compounding; Female; Glycolipids; Host-Pathogen Interactions; Immunogenicity, Vaccine; Lipid A; Liposomes; Lung; Mice, Inbred C57BL; Mycobacterium tuberculosis; Quaternary Ammonium Compounds; Receptors, Pattern Recognition; Time Factors; Tuberculosis Vaccines; Tuberculosis, Pulmonary; Vaccination; Vaccines, Subunit; Virulence

There is a link between high lipopolysaccharide (LPS) levels in the blood and the metabolic syndrome, and metabolic syndrome predisposes patients to severe COVID-19. Here, we define an interaction between SARS-CoV-2 spike (S) protein and LPS, leading to aggravated inflammation in vitro and in vivo. Native gel electrophoresis demonstrated that SARS-CoV-2 S protein binds to LPS. Microscale thermophoresis yielded a KD of ∼47 nM for the interaction. Computational modeling and all-atom molecular dynamics simulations further substantiated the experimental results, identifying a main LPS-binding site in SARS-CoV-2 S protein. S protein, when combined with low levels of LPS, boosted nuclear factor-kappa B (NF-κB) activation in monocytic THP-1 cells and cytokine responses in human blood and peripheral blood mononuclear cells, respectively. The in vitro inflammatory response was further validated by employing NF-κB reporter mice and in vivo bioimaging. Dynamic light scattering, transmission electron microscopy, and LPS-FITC analyses demonstrated that S protein modulated the aggregation state of LPS, providing a molecular explanation for the observed boosting effect. Taken together, our results provide an interesting molecular link between excessive inflammation during infection with SARS-CoV-2 and comorbidities involving increased levels of bacterial endotoxins.

Topics: Animals; Binding Sites; COVID-19; Cytokine Release Syndrome; Disease Models, Animal; Gram-Negative Bacterial Infections; Humans; In Vitro Techniques; Inflammation; Lipid A; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Mice, Transgenic; Models, Immunological; Models, Molecular; Molecular Docking Simulation; Protein Binding; Protein Interaction Domains and Motifs; Respiratory Distress Syndrome; Risk Factors; SARS-CoV-2; Spike Glycoprotein, Coronavirus

Monophosphoryl lipid A (MPLA) is a TLR4 agonist that has potent immunomodulatory properties and modulates innate immune function to improve host resistance to infection with common nosocomial pathogens in mice. The goal of this study was to assess the safety and efficacy of MPLA in a sheep model of burn injury and Pseudomonas aeruginosa pneumonia. The sheep provides a favorable model for preclinical testing as their response to TLR4 agonists closely mimics that of humans.. Twelve chronically instrumented adult female Merino sheep received 20% total body surface area, third-degree cutaneous burn under anesthesia and analgesia. At 24 h after burn, sheep were randomly allocated to receive: MPLA (2.5 μg/kg i.v., n = 6), or vehicle (i.v., n = 6). At 24 h after MPLA or vehicle treatment, Pseudomonas aeruginosa pneumonia was induced. Sheep were mechanically ventilated, fluid resuscitated and cardiopulmonary variables were monitored for 24 h after induction of pneumonia. Cytokine production, vascular barrier function, and lung bacterial burden were also measured.. MPLA infusion induced small and transient alterations in core body temperature, heart rate, pulmonary artery pressure, and pulmonary vascular resistance. Pulmonary mechanics were not altered. Vehicle-treated sheep developed severe acute lung injury during Pseudomonas aeruginosa pneumonia, which was attenuated by MPLA as indicated by improved PaO2/FiO2 ratio, oxygenation index, and shunt fraction. Sheep treated with MPLA also exhibited less vascular leak, lower blood lactate levels, and lower modified organ injury score. MPLA treatment attenuated systemic cytokine production and decreased lung bacterial burden.. MPLA was well tolerated in burned sheep and attenuated development of acute lung injury, lactatemia, cytokinemia, vascular leak, and hemodynamic changes caused by Pseudomonas aeruginosa pneumonia.

Topics: Adjuvants, Immunologic; Animals; Burns; Disease Models, Animal; Female; Lipid A; Multiple Organ Failure; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Sheep

Topics: Allergens; Animals; Antigens, Plant; Cells, Cultured; Cryptomeria; Disease Models, Animal; Glucosides; Humans; Immunotherapy; Japan; Leukocytes, Mononuclear; Lipid A; Mice; Mice, Inbred BALB C; Plant Proteins; Pollen; Rhinitis, Allergic, Seasonal; Toll-Like Receptor 4; Treatment Outcome

Generated by gram-negative bacteria, lipopolysaccharides (LPSs) are one of the most abundant and potent immunomodulatory substances present in the intestinal lumen. Interaction of agonistic LPS with the host myeloid-differentiation-2/Toll-like receptor 4 (MD-2/TLR4) receptor complex results in nuclear factor κB (NF-κB) activation, followed by the robust induction of pro-inflammatory immune responses. Here we have isolated LPS from a common gut commensal, Bacteroides vulgatus mpk (BVMPK), which provides only weak agonistic activity. This weak agonistic activity leads to the amelioration of inflammatory immune responses in a mouse model for experimental colitis, and it was in sharp contrast to strong agonists and antagonists. In this context, the administration of BVMPK LPS into mice with severe intestinal inflammation re-established intestinal immune homeostasis within only 2 weeks, resulting in the clearance of all symptoms of inflammation. These inflammation-reducing properties of weak agonistic LPS are grounded in the induction of a special type of endotoxin tolerance via the MD-2/TLR4 receptor complex axis in intestinal lamina propria CD11c

Topics: Animals; Biomarkers; CD11c Antigen; Colitis; Disease Models, Animal; Gastrointestinal Microbiome; Homeostasis; Humans; Immunity, Mucosal; Inflammatory Bowel Diseases; Intestinal Mucosa; Lipid A; Lipopolysaccharides; Mice; Mice, Knockout; Positron-Emission Tomography

Topics: Animals; Antigens, Bacterial; BCG Vaccine; Beijing; Disease Models, Animal; Glucosides; Humans; Immunization, Secondary; Lipid A; Mice; Mycobacterium tuberculosis; Th1 Cells; Tuberculosis; Tuberculosis Vaccines; Vaccination

Colistin resistance in

Topics: Acinetobacter baumannii; Acinetobacter Infections; Animals; Anti-Bacterial Agents; Bacterial Proteins; Colistin; Disease Models, Animal; Drug Resistance, Bacterial; Humans; Lipid A; Microbial Sensitivity Tests; Moths; Transcription Factors

Ischaemic stroke is a leading cause of death, disability, and a high unmet medical need. Post-reperfusion inflammation and an up-regulation of toll-like receptor 4 (TLR4), an upstream sensor of innate immunity, are associated with poor outcome in stroke patients. Here, we identified the therapeutic effect of targeting the LPS/TLR4 signal transduction pathway.. We tested the effect of the TLR4 inhibitor, eritoran (E5564) in different in vitro ischaemia-related models: human organotypic cortex culture, rat organotypic hippocampal cultures, and primary mixed glia cultures. We explored the therapeutic window of E5564 in the transient middle cerebral artery occlusion model of cerebral ischaemia in mice.. In vivo, administration of E5564 1 and 4 hr post-ischaemia reduced the expression of different pro-inflammatory chemokines and cytokines, infarct volume, blood-brain barrier breakdown, and improved neuromotor function, an important clinically relevant outcome. In the human organotypic cortex culture, E5564 reduced the activation of microglia and ROS production evoked by LPS.. TLR4 signalling has a causal role in the inflammation associated with a poor post-stroke outcome. Importantly, its inhibition by eritoran (E5564) provides neuroprotection both in vitro and in vivo, including in human tissue, suggesting a promising new therapeutic approach for ischaemic stroke.

Topics: Animals; Anti-Inflammatory Agents; Brain; Brain Ischemia; Cell Line; Cytokines; Disease Models, Animal; Female; Humans; Lipid A; Male; Mice, Inbred C57BL; Microglia; Neuroprotective Agents; Phenotype; Rats, Sprague-Dawley; Reactive Oxygen Species; Toll-Like Receptor 4

LPS inhibits

Topics: Adaptor Proteins, Vesicular Transport; Adjuvants, Immunologic; Animals; Bicarbonates; Cytoprotection; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Interleukin-1 Receptor-Associated Kinases; Intracellular Signaling Peptides and Proteins; Lipid A; Loop of Henle; Mice, Inbred C57BL; Mice, Knockout; Myeloid Differentiation Factor 88; Phosphatidylinositol 3-Kinase; Phosphorylation; Rats, Sprague-Dawley; Renal Reabsorption; Sepsis; Signal Transduction; Toll-Like Receptor 4

The small intestine is one of the most sensitive organs to irradiation injury, and the development of high effective radioprotectants especially with low toxicity for intestinal radiation sickness is urgently needed. Monophosphoryl lipid A (MPLA) was found to be radioprotective in our previous study, while its effect against the intestinal radiation injury remained unknown. In the present study, we firstly determined the intestinal apoptosis after irradiation injury according to the TUNEL assay. Subsequently, we adopted the immunofluorescence technique to assess the expression levels of different biomarkers including Ki67,

Topics: Animals; Apoptosis; Cytokines; Disease Models, Animal; DNA Damage; Enteritis; Inflammation Mediators; Intestinal Mucosa; Lipid A; Mice; Mice, Knockout; Radiation Injuries, Experimental; Radiation, Ionizing; Toll-Like Receptor 4

Allergen-specific immunotherapy (SIT) effectively alleviates type I allergic diseases characterized by T helper (Th)2-type immunity. Our recent studies have shown that a synthetic trivalent glycocluster, triacedimannose (TADM), suppresses the Th2-type allergic inflammation. The aim of this study was to compare TADM with two well-known adjuvants, unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG) and monophosphoryl lipid A (MPLA) in a grass allergen-induced chronic allergic inflammation model in mice.. Female BALB/c mice were intranasally sensitized with 50 μL of timothy grass pollen extract (TE) twice a week for a period of 15 weeks. Therapeutic intranasal treatments were then performed once a week after the tenth intranasal TE instillation using TADM (10 or 25 μg/50 μL), CpG-ODN (20 μg/50 μL) or MPLA (2 μg/50 μL). Groups of 9-10 animals per treatment were killed 24 hours after the last timothy dosage. Blood, bronchoalveolar lavage (BAL) fluids and lung biopsies were taken for subsequent analysis.. When mice were repeatedly exposed to TE for 15 weeks, the number of eosinophils and lymphocytes increased in the BAL fluids. The eosinophil and lymphocyte counts decreased dose-dependently and were practically abolished in the mice treated with TADM. Treatments with MPLA or CpG significantly increased the numbers of neutrophils, while CpG nonsignificantly decreased eosinophilia compared to timothy exposure.. A novel synthetic glycocluster molecule inhibited the development of grass-induced eosinophilic pulmonary inflammation in mice when administrated in the airways. This compound could be a candidate to be used either as an adjuvant in SIT or as a topical anti-inflammatory treatment.

Topics: Adjuvants, Immunologic; Allergens; Animals; Bronchoalveolar Lavage Fluid; Desensitization, Immunologic; Disaccharides; Disease Models, Animal; Eosinophils; Female; Hypersensitivity; Lipid A; Lymphocyte Count; Mannans; Mice; Mice, Inbred BALB C; Oligodeoxyribonucleotides; Phleum; Plant Extracts; Pneumonia; Pollen; Statistics, Nonparametric

Finding a neuroprotective strategy to rescue patients suffering from acute brain damage is of great interest. Monophosphoryl lipid A (MPL) is a derivative of lipopolysaccharide (LPS) that lacks many of the endotoxic properties of the parent molecule, and yet has similar protective effect. Here, we report the first evidence that MPL preconditioning, similar to LPS preconditioning, can induce neuroprotection against cerebral ischemia. MPL (0.5, 1, 5 μg/rat) was injected unilaterally into the left cerebral ventricle of male rats, and 48 h later, rats were subjected to ipsilateral selective hippocampal ischemia using a modified version of the photothrombotic method. The neuroprotective effects of MPL and LPS were evaluated by measuring infarct size and assessing cognitive function. The expression level of some inflammatory and anti-inflammatory cytokines involving in TLR4 signaling pathway was also measured. Cognitive impairment and infarct size were obvious in control group receiving normal saline intracerebroventricularly and then selective hippocampal ischemia, compared to the sham group. Immunologic preconditioning with MPL or LPS significantly reduced infarct size and improved cognitive function. Additionally, immunologic preconditioning resulted in inflammatory mediators, NF-κB and TNF-α, down-regulation but anti-inflammatory mediators, IRF3, IFN-β, and TGF-β, up-regulation. Our data showed that both MPL and LPS preconditioning may reprogram the TLR4 signaling pathway to produce a cytokine profile which eventually leads to neuroprotection against ischemia injury. MPL, unlike LPS, is safe and well tolerated in clinic, thus it could be considered as a new approach in prevention or even treatment of cerebral ischemic insult consequences.

Topics: Animals; Avoidance Learning; Brain Ischemia; Cognitive Dysfunction; Disease Models, Animal; Dose-Response Relationship, Drug; Functional Laterality; Hippocampus; Lipid A; Male; Maze Learning; Neuroprotective Agents; NF-kappa B; Random Allocation; Rats, Wistar; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Metabolic acidosis is the most common acid-base disorder in septic patients and is associated with increased mortality. Previously, we demonstrated that sepsis induced by cecal ligation and puncture (CLP) impairs [Formula: see text] absorption in the medullary thick ascending limb (MTAL) by 1) decreasing the intrinsic [Formula: see text] absorptive capacity and 2) enhancing inhibition of [Formula: see text] absorption by LPS through upregulation of Toll-like receptor (TLR) 4 signaling. Both effects depend on ERK activation. Monophosphoryl lipid A (MPLA) is a detoxified TLR4 agonist that enhances innate antimicrobial immunity and improves survival following sepsis. Pretreatment of MTALs with MPLA in vitro prevents LPS inhibition of [Formula: see text] absorption. Here we examined whether pretreatment with MPLA would protect the MTAL against sepsis. Vehicle or MPLA was administered to mice 48 h before sham or CLP surgery, and MTALs were studied in vitro 18 h postsurgery. Pretreatment with MPLA prevented the effects of sepsis to decrease the basal [Formula: see text] absorption rate and enhance inhibition by LPS. These protective effects were mediated through MPLA stimulation of a Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β-(TRIF)-dependent phosphatidylinositol 3-kinase-Akt pathway that prevents sepsis- and LPS-induced ERK activation. The effects of MPLA to improve MTAL [Formula: see text] absorption were associated with marked improvement in plasma [Formula: see text] concentration, supporting a role for the kidneys in the pathogenesis of sepsis-induced metabolic acidosis. These studies support detoxified TLR4-based immunomodulators, such as MPLA, that enhance antimicrobial responses as a safe and effective approach to prevent or treat sepsis-induced renal tubule dysfunction and identify cell signaling pathways that can be targeted to preserve MTAL [Formula: see text] absorption and attenuate metabolic acidosis during sepsis.

Topics: Acid-Base Equilibrium; Acidosis; Adaptor Proteins, Vesicular Transport; Animals; Bicarbonates; Disease Models, Animal; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Lipid A; Loop of Henle; Male; Mice, Inbred C57BL; Mice, Knockout; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Renal Reabsorption; Sepsis; Signal Transduction; Toll-Like Receptor 4

Annual vaccination is not effective in conferring cross-protection against antigenically different influenza viruses. Therefore, it is of high priority to improve the cross protective efficacy of influenza vaccines. We investigated the adjuvant effects of monophosphoryl lipid A (MPL) and oligodeoxynucleotide CpG (CpG) on promoting homologous protection and cross-protection after vaccination of C57BL/6 and BALB/c mice with inactivated split virus. Combination adjuvant effects of MPL and CpG on improving homologous and cross protective vaccine efficacy were evident as shown by higher levels of homologous and cross-reactive binding IgG and hemagglutination inhibiting antibodies. Combination adjuvant effects on enhancing the protective efficacy against homologous and heterosubtypic virus were demonstrated by less weight loss, lower airway inflammatory disease, and better control of viral loads as well as prevention of inflammatory cytokines and cellular infiltrates. Overall, the findings in this study suggest that a combination adjuvant of different toll-like receptor ligands exhibits a unique pattern of innate and adaptive immune responses, contributing to improved homologous and heterosubtypic cross-protection by inactivated split virion influenza vaccination.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Viral; Body Weight; Cross Protection; Cytokines; Disease Models, Animal; Humans; Immunoglobulin G; Influenza Vaccines; Influenza, Human; Lipid A; Mice, Inbred BALB C; Mice, Inbred C57BL; Oligodeoxyribonucleotides; Vaccines, Inactivated; Vaccines, Subunit; Viral Load

To compare the immunological and clinical changes induced by allergen-specific immunotherapy (AIT) using different adjuvants.. Olea europaea pollen-sensitized mice were treated with olea plus aluminum hydroxide, calcium phosphate, monophosphoryl lipid A (MPL) or immunostimulatory sequences (ISS).. Aluminum hydroxide seems to drive initially to a Th2-type response. Bacteria-derived adjuvants (MPL and ISS) skew the immune response toward Th1 and Treg pathways. Specific-IgE production was lower after AIT with MPL and ISS. Moreover, IgG2a production significantly increased in ISS-treated mice. Bacteria-derived adjuvants also improved the Th1 cytokine response due to IFN-γ higher secretion. In addition, they improved bronchial hyper-reactivity and lung inflammation.. Bacteria-derived adjuvants may enhance the efficacy of AIT.

Topics: Adjuvants, Immunologic; Allergens; Aluminum Hydroxide; Animals; Antigens, Plant; Calcium Phosphates; Desensitization, Immunologic; Disease Models, Animal; Female; Humans; Hypersensitivity; Lipid A; Mice; Mice, Inbred BALB C; Olea; Plant Extracts; Pollen; Respiratory Function Tests

One strategy to control leishmaniasis is vaccination with potent antigens alongside suitable adjuvants. The use of toll-like receptor (TLR) agonists as adjuvants is a promising approach in Leishmania vaccine research. Leishmania (L.) tropica is among the less-investigated Leishmania species and a causative agent of cutaneous and sometimes visceral leishmaniasis with no approved vaccine against it. In the present study, we assessed the adjuvant effects of a TLR4 agonist, monophosphoryl lipid A (MPL) and a TLR7/8 agonist, R848 beside two different types of Leishmania vaccine candidates; namely, whole-cell soluble L. tropica antigen (SLA) and recombinant L. tropica stress-inducible protein-1 (LtSTI1). BALB/c mice were vaccinated three times by the antigens (SLA or LtSTI1) with MPL or R848 and then were challenged by L. tropica. Delayed-type hypersensitivity (DTH), parasite load, disease progression and cytokines (IL-10 and IFN-γ) responses were assessed. In general compared to SLA, application of LtSTI1 resulted in higher DTH, higher IFN-γ response and lower lymph node parasite load. Also compared to R848, MPL as an adjuvant resulted in higher DTH and lower lymph node parasite load. Although, no outstanding ability for SLA and R848 in evoking immune responses of BALB/c mice against L. tropica infection could be observed, our data suggest that LtSTI1 and MPL have a better potential to control L. tropica infection and could be pursued for the development of effective vaccination strategies.

Topics: Adjuvants, Immunologic; Animals; Antigens, Protozoan; Cytokines; Disease Models, Animal; Female; Imidazoles; Leishmania tropica; Leishmaniasis; Leishmaniasis Vaccines; Lipid A; Mice, Inbred BALB C; Parasite Load; Protozoan Proteins; Random Allocation; Recombinant Proteins; Toll-Like Receptors; Vaccination; Vaccines, Inactivated

Bacterial infection of the kidney leads to a rapid cascade of host protective responses, many of which are still poorly understood. We have previously shown that following kidney infection with uropathogenic Escherichia coli (UPEC), vascular coagulation is quickly initiated in local perivascular capillaries that protects the host from progressing from a local infection to systemic sepsis. The signaling mechanisms behind this response have not however been described. In this study, we use a number of in vitro and in vivo techniques, including intravital microscopy, to identify two previously unrecognized components influencing this protective coagulation response. The acylation state of the Lipid A of UPEC lipopolysaccharide (LPS) is shown to alter the kinetics of local coagulation onset in vivo. We also identify epithelial CD147 as a potential host factor influencing infection-mediated coagulation. CD147 is expressed by renal proximal epithelial cells infected with UPEC, contingent to bacterial expression of the α-hemolysin toxin. The epithelial CD147 subsequently can activate tissue factor on endothelial cells, a primary step in the coagulation cascade. This study emphasizes the rapid, multifaceted response of the kidney tissue to bacterial infection and the interplay between host and pathogen during the early hours of renal infection.

Topics: Animals; Bacterial Infections; Basigin; Biomarkers; Blood Coagulation; Cell Line; Cytokines; Disease Models, Animal; Epithelial Cells; Humans; Inflammation Mediators; Lipid A; Male; Nephritis; Proteome; Proteomics; Rats; Signal Transduction

Malaria caused by Plasmodium falciparum continues to threaten millions of people living in the tropical parts of the world. A vaccine that confers sterile and life-long protection remains elusive despite more than 30years of effort and resources invested in solving this problem. Antibodies to a malaria vaccine candidate circumsporozoite protein (CSP) can block invasion and can protect humans against malaria. We have manufactured the Falciparum Malaria Protein-013 (FMP013) vaccine based on the nearly full-length P. falciparum CSP 3D7 strain sequence. We report here immunogenicity and challenge data on FMP013 antigen in C57BL/6 mice formulated with two novel adjuvants of the Army Liposome Formulation (ALF) series and a commercially available adjuvant Montanide ISA 720 (Montanide) as a control. ALF is a liposomal adjuvant containing a synthetic monophosphoryl lipid A (3D-PHAD®). In our study, FMP013 was adjuvanted with ALF alone, ALF containing aluminum hydroxide (ALFA) or ALF containing QS-21 (ALFQ). Adjuvants ALF and ALFA induced similar antibody titers and protection against transgenic parasite challenge that were comparable to Montanide. ALFQ was superior to the other three adjuvants as it induced higher antibody titers with improved boosting after the third immunization, higher serum IgG2c titers, and enhanced protection. FMP013+ALFQ also augmented the numbers of splenic germinal center-derived activated B-cells and antibody secreting cells compared to Montanide. Further, FMP013+ALFQ induced antigen-specific IFN-γ ELISPOT activity, CD4

Topics: Adjuvants, Immunologic; Animals; Antibodies, Protozoan; B-Lymphocytes; CD4-Positive T-Lymphocytes; Disease Models, Animal; Enzyme-Linked Immunospot Assay; Female; Interferon-gamma; Lipid A; Liposomes; Malaria Vaccines; Malaria, Falciparum; Mice, Inbred C57BL; Protozoan Proteins; Saponins

Topics: Acyltransferases; Animals; Bacterial Proteins; Cells, Cultured; Disease Models, Animal; Gene Deletion; Gene Expression Regulation, Bacterial; Klebsiella Infections; Klebsiella pneumoniae; Lepidoptera; Lipid A; Macrophages; Mass Spectrometry; Mice; Phagocytosis; Virulence; Virulence Factors

Alpha-synuclein (α-syn) aggregation represents the pathological hallmark of α-synucleinopathies like Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Toll-like receptors (TLRs) are a family of highly conserved molecules that recognize pathogen-associated molecular patterns and define the innate immunity response. It was previously shown that TLR4 plays a role in the clearance of α-syn, suggesting that TLR4 up-regulation in microglia may be a natural mechanism to improve the clearance of α-syn. However, administration of TLR4 ligands could also lead to dangerous adverse effects associated with the induction of toxic inflammatory responses. Monophosphoryl lipid A (MPLA) is a TLR4 selective agonist and a potent inducer of phagocytosis which does not trigger strong toxic inflammatory responses as compared to lipopolysaccharide (LPS). We hypothesize that MPLA treatment will lead to increased clearance of α-syn inclusions in the brain of transgenic mice overexpressing α-syn in oligodendrocytes under the proteolipid protein promoter (PLP-α-syn mouse model of MSA), without triggering toxic cytokine release, thus leading to a general amelioration of the pathology.. Six month old PLP-α-syn mice were randomly allocated to four groups and received weekly intraperitoneal injections of MPLA (50 or 100 μg), LPS or vehicle. After a 12-week treatment period, motor behavior was assessed with the pole test. Brains and plasma samples were collected for neuropathological and immunological analysis.. Chronic systemic MPLA treatment of PLP-α-syn mice led to increased uptake of α-syn by microglial cells, a significant motor improvement, rescue of nigral dopaminergic and striatal neurons and region-specific reduction of the density of oligodendroglial α-syn cytoplasmic inclusions in the absence of a marked systemic inflammatory response.. Our findings demonstrate beneficial effects of chronic MPLA treatment in transgenic PLP-α-syn mice. MPLA appears to be an attractive therapeutic candidate for disease modification trials in MSA and related α-synucleinopathies.

Topics: alpha-Synuclein; Animals; Disease Models, Animal; Female; Lipid A; Male; Mice, Transgenic; Multiple System Atrophy; Neurons; Parkinson Disease; Substantia Nigra; Toll-Like Receptor 4

The infectious diseases caused by multidrug-resistant bacteria pose serious threats to humankind. It has been suggested that an antibiotic targeting LpxC of the lipid A biosynthetic pathway in Gram-negative bacteria is a promising strategy for curing Gram-negative bacterial infections. However, experimental proof of this concept is lacking. Here, we describe our discovery and characterization of a biphenylacetylene-based inhibitor of LpxC, an essential enzyme in the biosynthesis of the lipid A component of the outer membrane of Gram-negative bacteria. The compound LPC-069 has no known adverse effects in mice and is effective

Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; Benzamides; Disease Models, Animal; Drug Resistance, Multiple, Bacterial; Enzyme Inhibitors; Female; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Lipid A; Mice; Morpholines; Plague; Yersinia pestis

An efficient method was developed for the synthesis of a GM2 derivative suitable for the conjugation with various biomolecules. This GM2 derivative was covalently linked to keyhole limpet hemocyanin (KLH) and monophosphoryl lipid A (MPLA) to form novel therapeutic cancer vaccines. Immunological evaluations of the resultant conjugates in mice revealed that they elicited robust GM2-specific overall and IgG antibody responses. Moreover, the GM2-MPLA conjugate was disclosed to elicit strong immune responses without the use of an adjuvant, proving its self-adjuvant property. The antisera of both conjugates showed strong binding and mediated similarly effective complement-dependent cytotoxicity to GM2-expressing cancer cell line MCF-7. Based on these results, it was concluded that both GM2-MPLA and GM2-KLH are promising candidates as therapeutic cancer vaccines, whereas fully synthetic GM2-MPLA, which has homogeneous and well-defined structure and self-adjuvant property, deserves more attention and studies.

Topics: Adjuvants, Immunologic; Animals; Antibody-Dependent Cell Cytotoxicity; Cancer Vaccines; Complement System Proteins; Disease Models, Animal; Female; G(M2) Ganglioside; Lipid A; Mice; Molecular Structure; Neoplasms; Vaccines, Synthetic; Xenograft Model Antitumor Assays

The lack of antifungals with low toxicity and short-term therapy for patients with paracoccidioidomycosis (PCM) led us to evaluate adjuvants in immunotherapeutic intervention. We have previously shown complete Freund's adjuvant (CFA) to be therapeutic on experimental PCM. Owing to CFA toxicity, here we tested adjuvants approved for clinical use or in preclinical phase in experimental mouse PCM. Of all, only monophosporyl lipid A (MPLA) demonstrates a beneficial effect, by reducing the fungal burden and increasing the concentrations of IFN-γ and TNF-α, which are immunoprotective in PCM. These results suggest that MPLA might improve intervention in PCM.

Topics: Animals; Disease Models, Animal; Immunologic Factors; Lipid A; Male; Mice, Inbred BALB C; Paracoccidioidomycosis; Therapeutic Uses; Treatment Outcome

Influenza is an acute respiratory disease and a major health problem worldwide. Since mucosal immunity plays a critical role in protection against influenza virus infection, mucosal immunization is considered a promising vaccination route. However, except for live-attenuated vaccines, there are no effective killed or recombinant mucosal influenza vaccines to date. Outer membrane vesicles (OMVs) are nano-sized vesicles produced by gram-negative bacteria, and contain various bacterial components capable of stimulating the immune system of the host. We generated an OMV with low endotoxicity (fmOMV) by modifying the structure of the lipid A moiety of lipopolysaccharide and investigated its effect as an intranasal vaccine adjuvant in an influenza vaccine model. In this model, fmOMV exhibited reduced toll-like receptor 4-stimulating activity and attenuated endotoxicity compared to that of native OMV. Intranasal injection of the vaccine antigen with fmOMV significantly increased systemic antibody and T cell responses, mucosal IgA levels, and the frequency of lung-resident influenza-specific T cells. In addition, the number of antigen-bearing CD103

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Antibodies, Viral; Dendritic Cells; Disease Models, Animal; Extracellular Vesicles; Female; Immunoglobulin A; Influenza Vaccines; Lipid A; Lymph Nodes; Mice, Inbred C57BL; Mucous Membrane; Orthomyxoviridae; Orthomyxoviridae Infections; Survival Analysis; T-Lymphocytes; Treatment Outcome

Respiratory syncytial virus (RSV) infection of children previously immunized with a nonlive, formalin-inactivated (FI)-RSV vaccine has been associated with serious enhanced respiratory disease (ERD). Consequently, detailed studies of potential ERD are a critical step in the development of nonlive RSV vaccines targeting RSV-naive children and infants. The fusion glycoprotein (F) of RSV in either its postfusion (post-F) or prefusion (pre-F) conformation is a target for neutralizing antibodies and therefore an attractive antigen candidate for a pediatric RSV subunit vaccine. Here, we report the evaluation of RSV post-F and pre-F in combination with glucopyranosyl lipid A (GLA) integrated into stable emulsion (SE) (GLA-SE) and alum adjuvants in the cotton rat model. Immunization with optimal doses of RSV F antigens in the presence of GLA-SE induced high titers of virus-neutralizing antibodies and conferred complete lung protection from virus challenge, with no ERD signs in the form of alveolitis. To mimic a waning immune response, and to assess priming for ERD under suboptimal conditions, an antigen dose de-escalation study was performed in the presence of either GLA-SE or alum. At low RSV F doses, alveolitis-associated histopathology was unexpectedly observed with either adjuvant at levels comparable to FI-RSV-immunized controls. This occurred despite neutralizing-antibody titers above the minimum levels required for protection and with no/low virus replication in the lungs. These results emphasize the need to investigate a pediatric RSV vaccine candidate carefully for priming of ERD over a wide dose range, even in the presence of strong neutralizing activity, Th1 bias-inducing adjuvant, and protection from virus replication in the lower respiratory tract.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibodies, Neutralizing; Antibodies, Viral; Antibody-Dependent Enhancement; Disease Models, Animal; Lipid A; Recombinant Proteins; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus Vaccines; Sigmodontinae; Th1 Cells; Th2 Cells; Vaccines, Synthetic; Viral Fusion Proteins

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacterial Load; Bacterial Proteins; Colistin; Disease Models, Animal; Drug Resistance, Bacterial; Humans; Klebsiella Infections; Klebsiella pneumoniae; Lepidoptera; Lipid A; Lung; Membrane Proteins; Mice, Inbred C57BL; Polymyxins; Survival Analysis; Virulence

The magnitude of the LPS-elicited cytokine response is commonly used to assess immune function in critically ill patients. A suppressed response, known as endotoxin tolerance, is associated with worse outcomes, yet endotoxin tolerance-inducing TLR4 ligands are known to protect animals from infection. Thus, it remains unknown whether the magnitude of the LPS-elicited cytokine response provides an accurate assessment of antimicrobial immunity. To address this, the ability of diverse TLR ligands to modify the LPS-elicited cytokine response and resistance to infection were assessed. Priming of mice with LPS, monophosphoryl lipid A (MPLA), or poly(I:C) significantly reduced plasma LPS-elicited proinflammatory cytokines, reflecting endotoxin tolerance, whereas CpG-ODN-primed mice showed augmented cytokine production. In contrast, LPS, MPLA, and CpG-ODN, but not poly(I:C), improved the host response to a

Topics: Animals; Cytokines; Disease Models, Animal; Flow Cytometry; Ligands; Lipid A; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Poly I-C; Pseudomonas aeruginosa; Pseudomonas Infections; Toll-Like Receptors

Huanglian Jiedu Decoction (HJD), one of the classic recipes for relieving toxicity and fever, is a common method for treating sepsis in China. However, the effective components of HJD have not yet been identified. This experiment was carried out to elucidate the effective components of HJD against sepsis. Thus, seven fractions from HJD were tested using a biosensor to test their affinity for lipid A. The components obtained that had high lipid A-binding fractions were further separated, and their affinities to lipid A were assessed with the aid of a biosensor. The levels of LPS in the blood were measured, and pathology experiments were conducted. The LPS levels and mRNA expression analysis of TNF-α and IL-6 of the cell supernatant and animal tissue were evaluated to investigate the molecular mechanisms. Palmatine showed the highest affinity to lipid A and was evaluated by in vitro and in vivo experiments. The results of the in vitro and in vivo experiments indicated that the levels of LPS, TNF-α and IL-6 of the palmatine group were significantly lower than those of the sepsis model group (p<0.01). The group treated with palmatine showed strong neutralizing LPS activity in vivo. The palmatine group exhibited stronger protective activity on vital organs compared to the LPS-induced animal model. This verifies that HJD is a viable treatment option for sepsis given that there are multiple components in HJD that neutralize LPS, decrease the release of IL-6 and TNF-α induced by LPS, and protect vital organs.

Topics: Animals; Berberine Alkaloids; Disease Models, Animal; Drugs, Chinese Herbal; Gene Expression Regulation; Humans; Interleukin-6; Lipid A; Lipopolysaccharides; Mice; Mice, Inbred BALB C; RAW 264.7 Cells; Sepsis; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

There is currently no licensed prophylactic or therapeutic vaccine for HSV-2 infection.. We developed a novel preclinical vaccine candidate, G103, consisting of three recombinantly expressed HSV-2 proteins (gD and the UL19 and UL25 gene products) adjuvanted with the potent synthetic TLR4 agonist glucopyranosyl lipid A (GLA) formulated in stable emulsion. The vaccine was tested for immunogenicity and efficacy in pre-clinical models for preventative and therapeutic vaccination.. Vaccination of mice with G103 elicited antigen-specific binding and neutralizing antibody responses, as well as robust CD4 and CD8 effector and memory T cells. The T cell responses were further boosted by subsequent challenge with live virus. Prophylactic immunization completely protected against lethal intravaginal HSV-2 infection in mice, with only transient replication of virus in the genital mucosa and sterilizing immunity in dorsal root ganglia. Supporting the use of G103 therapeutically, the vaccine expanded both CD4 and CD8 T cells induced in mice by previous infection with HSV-2. In the guinea pig model of recurrent HSV-2 infection, therapeutic immunization with G103 was approximately 50% effective in reducing the number of lesions per animal as well as the overall lesions score.. Taken together, the data show that G103 is a viable candidate for development of a novel prophylactic and therapeutic HSV-2 vaccine.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Neutralizing; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Disease Models, Animal; Female; Glucosides; Guinea Pigs; Herpes Genitalis; Herpesvirus 2, Human; Herpesvirus Vaccines; Immunologic Memory; Lipid A; Mice, Inbred C57BL; Treatment Outcome; Vaccines, Subunit; Vaccines, Synthetic

Peripheral administration of lipopolysaccharide (LPS) elevates production of pro-inflammatory cytokines, and motivates the expression of sickness behaviors. In this study, we tested the ability of an LPS-derived adjuvant, monophosphoryl lipid A (MPLA), to prevent LPS-induced sickness behaviors in a burrowing paradigm. Testing occurred over a three-day period. Animals received a single injection of either MPLA or saline the first two days of testing. On day three, animals received either LPS or saline. Tissue from the dorsal hippocampus was collected for qRT-PCR to assess expression of IL-1β and IL-4. Results indicate that, during the pre-treatment phase, administration of MPLA induces an immune response sufficient to trigger sickness behaviors. However, we observed that animals pre-treated with MPLA for two days were resistant to LPS-induced sickness behaviors on day three. Results from the qRT-PCR analysis indicated that LPS-treated animals pre-treated with MPLA expressed significantly less IL-1β compared to LPS-treated animals pre-treated with saline. However, we did not observe a significant difference in IL-4 expression between groups. Therefore, results indicate that under the given parameters of the study, MPLA pre-treatment protects against LPS-induced sickness behaviors, at least in part, by decreasing expression of the pro-inflammatory cytokine IL-1β.

Topics: Adjuvants, Immunologic; Animals; Disease Models, Animal; Drug Administration Schedule; Gene Expression Regulation; Hippocampus; Illness Behavior; Interleukin-1beta; Interleukin-4; Lipid A; Lipopolysaccharides; Mice; Mice, Inbred C57BL; RNA, Messenger

Vibrio cholerae is the causative agent of cholera, an acute diarrheal disease that remains endemic in many parts of the world. The mechanisms underlying immunity to cholera remain poorly defined, though it is increasingly clear that protection is associated with antibodies against lipopolysaccharide (LPS). Here we report that ZAC-3, a monoclonal antibody against the core/lipid A region of V. cholerae LPS is a potent inhibitor of V. cholerae flagellum-based motility in viscous and liquid environments. ZAC-3 arrested motility of the classical Ogawa strain O395, as well as the El Tor Inaba strain C6706. In addition, we demonstrate, in the neonatal mouse model, that ZAC-3 IgG and Fab fragments significantly reduced the ability of both V. cholerae strains O395 and C6706 to colonize the intestinal epithelium, revealing the potential of antibodies against the core/lipid A to contribute to immunity across biotypes, possibly through a mechanism involving motility arrest.

Topics: Animals; Animals, Newborn; Antibodies, Bacterial; Antibodies, Monoclonal; Antigens, Bacterial; Bacterial Typing Techniques; Cholera; Disease Models, Animal; Flagella; Immunoglobulin Fab Fragments; Immunoglobulin G; Intestinal Mucosa; Lipid A; Mice; Movement; Vibrio cholerae

A combination therapy for combined injury (CI) using a non-specific immunomodulator, synthetic trehalose dicorynomycolate and monophosphoryl lipid A (STDCM-MPL), was evaluated to augment oral antimicrobial agents, levofloxacin (LVX) and amoxicillin (AMX), to eliminate endogenous sepsis and modulate cytokine production.. Female B6D2F(1)/J mice received 9.75 Gy cobalt-60 gamma-radiation and wound. Bacteria were isolated and identified in three tissues. Incidence of bacteria and cytokines were compared between treatment groups.. Results demonstrated that the lethal dose for 50% at 30 days (LD(50/30)) of B6D2F(1)/J mice was 9.42 Gy. Antimicrobial therapy increased survival in radiation-injured (RI) mice. Combination therapy increased survival after RI and extended survival time but did not increase survival after CI. Sepsis began five days earlier in CI mice than RI mice with Gram-negative species predominating early and Gram-positive species increasing later. LVX plus AMX eliminated sepsis in CI and RI mice. STDCM-MPL eliminated Gram-positive bacteria in CI and most RI mice but not Gram-negative. Treatments significantly modulated 12 cytokines tested, which pertain to wound healing or elimination of infection.. Combination therapy eliminates infection and prolongs survival time but does not assure CI mouse survival, suggesting that additional treatment for proliferative-cell recovery is required.

Topics: Amoxicillin; Animals; Anti-Infective Agents; Bacterial Infections; Chemokines; Cord Factors; Cytokines; Disease Models, Animal; Drug Therapy, Combination; Female; Gamma Rays; Growth Substances; Immunologic Factors; Levofloxacin; Lipid A; Mice; Radiation Injuries, Experimental; Sepsis; Skin; Wound Infection

Antibody-mediated capture of amyloid-beta (Aβ) in peripheral blood was identified as an attractive strategy to eliminate cerebral toxic amyloid in Alzheimer's disease (AD) patients and murine models. Alternatively, defective capacity of peripheral monocytes to engulf Aβ was reported in individuals with AD. In this report, we developed different approaches to investigate cellular uptake and phagocytosis of Aβ, and to examine how two immunological devices--an immunostimulatory Adjuvant System and different amyloid specific antibodies--may affect these biological events. Between one and thirteen months of age, APPswe X PS1.M146V (TASTPM) AD model mice had decreasing concentrations of Aβ in their plasma. In contrast, the proportion of blood monocytes containing Aβ tended to increase with age. Importantly, the TLR-agonist containing Adjuvant System AS01B primed monocytes to promote de novo Aβ uptake capacity, particularly in the presence of anti-Aβ antibodies. Biochemical experiments demonstrated that cells achieved Aβ uptake and internalization followed by Aβ degradation via mechanisms that required effective actin polymerization and proteolytic enzymes such as insulin-degrading enzyme. We further demonstrated that both Aβ-specific monoclonal antibodies and plasma from Aβ-immunized mice enhanced the phagocytosis of 1 μm Aβ-coated particles. Together, our data highlight a new biomarker testing to follow amyloid clearance within the blood and a mechanism of Aβ uptake by peripheral monocytes in the context of active or passive immunization, and emphasize on novel approaches to investigate this phenomenon.

Topics: Actins; Adjuvants, Immunologic; Alzheimer Disease; Amyloid beta-Peptides; Animals; Antibodies, Monoclonal; Brain; Cell Line; Disease Models, Animal; Drug Combinations; Immunophenotyping; Immunotherapy; Lipid A; Mice; Mice, Transgenic; Monocytes; Phagocytosis; Protein Multimerization; Proteolysis; Saponins; Vaccination

Monophosphoryl lipid A (MPLA), a nontoxic TLR4 ligand derived from lipopolysaccharide (LPS), is used clinically as an adjuvant in cancer, hepatitis, and malaria vaccines and in allergen-specific immunotherapy. Nevertheless, its cell-activating effects have not been analyzed in a comprehensive direct comparison including a wide range of different immune cells. Therefore, the objective of this study was the side-by-side comparison of the immune-modulating properties of MPLA and LPS on different immune cells.. Immune-activating properties of MPLA and LPS were compared in human monocytes and mast cells (MCs), a mouse endotoxin shock model (ESM), and mouse bone marrow (BM)-derived myeloid dendritic cells (mDCs), T cells (TCs), B cells, and MCs.. In a mouse in vivo ESM and a human ex vivo monocyte activation test (MAT), MPLA induced the same cytokine secretion pattern as LPS (ESM: IL-6, IL-12, TNF-α; MAT: IL-1β, IL-6, TNF-α), albeit at lower levels. Mouse mDCs and ex vivo isolated B cells stimulated with MPLA required a higher threshold to induce TRIF-dependent cytokine secretion (IL-1β, IL-6, IL-10, and TNF-α) than did LPS-stimulated cells. In mDC:DO11.10 CD4 TC cocultures, stimulation with MPLA, but not with LPS, resulted in enhanced OVA-specific IL-4 and IL-5 secretion from DO11.10 CD4 TCs. Unexpectedly, in both human and mouse MCs, MPLA, unlike LPS, did not elicit secretion of pro-inflammatory cytokines.. Compared to LPS, MPLA induced a qualitatively similar, but less potent pro-inflammatory immune response, but was unable to activate human or mouse MCs.

Topics: Adaptor Proteins, Vesicular Transport; Animals; Antigens; B-Lymphocytes; Cytokines; Dendritic Cells; Disease Models, Animal; Epithelial Cells; Humans; Inflammation Mediators; Lipid A; Lipopolysaccharides; Mast Cells; Mice; Mice, Knockout; Monocytes; Shock, Septic; Th2 Cells

Subunit vaccines based on the herpes simplex virus 2 (HSV-2) glycoprotein D (gD-2) have been the major focus of HSV-2 vaccine development for the past 2 decades. Based on the promising data generated in the guinea pig model, a formulation containing truncated gD-2, aluminum salt, and MPL (gD/AS04) advanced to clinical trials. The results of these trials, however, were unexpected, as the vaccine protected against HSV-1 infection but not against HSV-2. To address this discrepancy, we developed a Depot medroxyprogesterone acetate (DMPA)-treated cotton rat Sigmodon hispidus model of HSV-2 and HSV-1 genital infection. The severity of HSV-1 genital herpes was less than that of HSV-2 genital herpes in cotton rats, and yet the model allowed for comparative evaluation of gD/AS04 immunogenicity and efficacy. Cotton rats were intramuscularly vaccinated using a prime boost strategy with gD/AS04 (Simplirix vaccine) or control vaccine formulation (hepatitis B vaccine FENDrix) and subsequently challenged intravaginally with HSV-2 or HSV-1. The gD/AS04 vaccine was immunogenic in cotton rats and induced serum IgG directed against gD-2 and serum HSV-2 neutralizing antibodies but failed to efficiently protect against HSV-2 disease or to decrease the HSV-2 viral load. However, gD/AS04 significantly reduced vaginal titers of HSV-1 and better protected animals against HSV-1 compared to HSV-2 genital disease. The latter finding is generally consistent with the clinical outcome of the Herpevac trial of Simplirix. Passive transfer of serum from gD/AS04-immunized cotton rats conferred stronger protection against HSV-1 genital disease. These findings suggest the need for alternative vaccine strategies and the identification of new correlates of protection.. In spite of the high health burden of genital herpes, there is still no effective intervention against the disease. The significant gap in knowledge on genital herpes pathogenesis has been further highlighted by the recent failure of GSK HSV-2 vaccine Simplirix (gD/AS04) to protect humans against HSV-2 and the surprising finding that the vaccine protected against HSV-1 genital herpes instead. In this study, we report that gD/AS04 has higher efficacy against HSV-1 compared to HSV-2 genital herpes in the novel DMPA-synchronized cotton rat model of HSV-1 and HSV-2 infection. The findings help explain the results of the Simplirix trial.

Topics: Aluminum Hydroxide; Aniline Compounds; Animals; Disease Models, Animal; Drug Evaluation, Preclinical; Enzyme-Linked Immunosorbent Assay; Female; Herpes Genitalis; Herpesvirus 1, Human; Herpesvirus 2, Human; Injections, Intramuscular; Lipid A; Sigmodontinae; Vaccines, Subunit; Viral Envelope Proteins; Viral Vaccines

The Tc24 calcium binding protein from the flagellar pocket of Trypanosoma cruzi is under evaluation as a candidate vaccine antigen against Chagas disease. Previously, a DNA vaccine encoding Tc24 was shown to be an effective vaccine (both as a preventive and therapeutic intervention) in mice and dogs, as evidenced by reductions in T. cruzi parasitemia and cardiac amastigotes, as well as reduced cardiac inflammation and increased host survival. Here we developed a suitable platform for the large scale production of recombinant Tc24 (rTc24) and show that when rTc24 is combined with a monophosphoryl-lipid A (MPLA) adjuvant, the formulated vaccine induces a Th1-biased immune response in mice, comprised of elevated IgG2a antibody levels and interferon-gamma levels from splenocytes, compared to controls. These immune responses also resulted in statistically significant decreased T. cruzi parasitemia and cardiac amastigotes, as well as increased survival following T. cruzi challenge infections, compared to controls. Partial protective efficacy was shown regardless of whether the antigen was expressed in Escherichia coli or in yeast (Pichia pastoris). While mouse vaccinations will require further modifications in order to optimize protective efficacy, such studies provide a basis for further evaluations of vaccines comprised of rTc24, together with alternative adjuvants and additional recombinant antigens.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Protozoan; Antigens, Protozoan; Chagas Disease; Cloning, Molecular; Disease Models, Animal; Escherichia coli; Female; Gene Expression; Interferon-gamma; Leukocytes, Mononuclear; Lipid A; Mice, Inbred BALB C; Parasite Load; Parasitemia; Pichia; Protozoan Vaccines; Recombinant Proteins; Spleen; Survival Analysis; Th1 Cells; Trypanosoma cruzi; Vaccines, Synthetic

Neisseria gonorrhoeae causes gonorrhea, a sexually transmitted infection characterized by inflammation of the cervix or urethra. However, a significant subset of patients with N. gonorrhoeae remain asymptomatic, without evidence of localized inflammation. Inflammatory responses to N. gonorrhoeae are generated by host innate immune recognition of N. gonorrhoeae by several innate immune signaling pathways, including lipooligosaccharide (LOS) and other pathogen-derived molecules through activation of innate immune signaling systems, including toll-like receptor 4 (TLR4) and the interleukin-1β (IL-1β) processing complex known as the inflammasome. The lipooligosaccharide of N. gonorrhoeae has a hexa-acylated lipid A. N. gonorrhoeae strains that carry an inactivated msbB (also known as lpxL1) gene produce a penta-acylated lipid A and exhibit reduced biofilm formation, survival in epithelial cells, and induction of epithelial cell inflammatory signaling. We now show that msbB-deficient N. gonorrhoeae induces less inflammatory signaling in human monocytic cell lines and murine macrophages than the parent organism. The penta-acylated LOS exhibits reduced toll-like receptor 4 signaling but does not affect N. gonorrhoeae-mediated activation of the inflammasome. We demonstrate that N. gonorrhoeae msbB is dispensable for initiating and maintaining infection in a murine model of gonorrhea. Interestingly, infection with msbB-deficient N. gonorrhoeae is associated with less localized inflammation. Combined, these data suggest that TLR4-mediated recognition of N. gonorrhoeae LOS plays an important role in the pathogenesis of symptomatic gonorrhea infection and that alterations in lipid A biosynthesis may play a role in determining symptomatic and asymptomatic infections.

Topics: Acylation; Acyltransferases; Analysis of Variance; Animals; Caspase 1; Cells, Cultured; Chemokines; Cytokines; Disease Models, Animal; Female; Gonorrhea; Humans; Inflammation; Lipid A; Lipopolysaccharides; Macrophages; Mice; Mice, Inbred BALB C; Monocytes; Neisseria gonorrhoeae; Signal Transduction

The development of adjuvants for vaccines has become an important area of research as the number of protein-based vaccines against infectious pathogens increases. Currently, there are a number of adjuvant-based Mycobacterium tuberculosis vaccines in clinical trials that have shown efficacy in animal models. Despite these novel adjuvants, there is still a need to design new and more versatile adjuvants that have minimal adverse side effects but produce robust long-lasting adaptive immune responses. To this end, we hypothesized that Bacillus subtilis spores may provide the appropriate innate signals that are required to generate such vaccine-mediated responses, which would be sufficient to reduce the mycobacterial burden after infection with M. tuberculosis. In addition, we compared the response generated by B. subtilis spores to that generated by monophosphoryl lipid A (MPL), which has been used extensively to test tuberculosis vaccines. The well-characterized, 6-kDa early secretory antigenic target of M. tuberculosis (ESAT-6; Rv3875) was used as a test antigen to determine the T cell activation potential of each adjuvant. Inoculated into mice, B. subtilis spores induced a strong proinflammatory response and Th1 immunity, similar to MPL; however, unlike MPL formulated with dimethyldioctadecylammonium (DDA) bromide, it failed to induce significant levels of interleukin-17A (IL-17A) and was unable to significantly reduce the mycobacterial burden after pulmonary infection with M. tuberculosis. Further analysis of the activity of MPL-DDA suggested that IL-17A was required for protective immunity. Taken together, the data emphasize the requirement for a network of cytokines that are essential for protective immunity.

Topics: Adjuvants, Immunologic; Animals; Bacillus subtilis; Bacterial Load; Disease Models, Animal; Female; Interferon-gamma; Interleukin-17; Lipid A; Lung; Mice, Inbred C57BL; Mycobacterium tuberculosis; Spores, Bacterial; T-Lymphocytes; Tuberculosis; Tuberculosis Vaccines

Pathogen transmission cycles require many steps: initial colonization, growth and persistence, shedding, and transmission to new hosts. Alterations in the membrane components of the bacteria, including lipid A, the membrane anchor of lipopolysaccharide, could affect any of these steps via its structural role protecting bacteria from host innate immune defenses, including antimicrobial peptides and signaling through Toll-like receptor 4 (TLR4). To date, lipid A has been shown to affect only the within-host dynamics of infection, not the between-host dynamics of transmission. Here, we investigate the effects of lipid A modification in a mouse infection and transmission model. Disruption of the Bordetella bronchiseptica locus (BB4268) revealed that ArnT is required for addition of glucosamine (GlcN) to B. bronchiseptica lipid A. ArnT modification of lipid A did not change its TLR4 agonist activity in J774 cells, but deleting arnT decreased resistance to killing by cationic antimicrobial peptides, such as polymyxin B and β-defensins. In the standard infection model, mutation of arnT did not affect B. bronchiseptica colonization, growth, persistence throughout the respiratory tract, recruitment of neutrophils to the nasal cavity, or shedding of the pathogen. However, the number of bacteria necessary to colonize a host (50% infective dose [ID50]) was 5-fold higher for the arnT mutant. Furthermore, the arnT mutant was defective in transmission between hosts. These results reveal novel functions of the ArnT lipid A modification and highlight the sensitivity of low-dose infections and transmission experiments for illuminating aspects of infectious diseases between hosts. Factors such as ArnT can have important effects on the burden of disease and are potential targets for interventions that can interrupt transmission.

Topics: Animals; Antimicrobial Cationic Peptides; Bordetella bronchiseptica; Bordetella Infections; Disease Models, Animal; Glucosamine; Hexosyltransferases; Lipid A; Mice; Mice, Inbred C57BL; Microbial Viability

Periodontitis is a disease of polymicrobial etiology characterized by inflammation, degradation of host tissue, and bone that irreversibly destroys the supporting apparatus of teeth. Porphyromonas gingivalis contains lipid A with structural heterogeneity that has been postulated to contribute to the initiation of dysbiosis in oral communities by modulating the host response, thereby creating a permissive environment for its growth. We examined two P. gingivalis lipid A phosphatase mutants which contain different "locked" lipid A structures that induce different host cellular responses for their ability to induce dysbiosis and periodontitis in rabbits. Lipopolysaccharide (LPS) preparations obtained from these strains were also examined. After repeated applications of all strains and their respective LPS preparations, P. gingivalis wild type, but not the lipid A mutants, had a significant impact on both the oral commensal microbial load and composition. In contrast, in rabbits exposed to the mutant strains or the LPS preparations, the microbial load did not increase, and yet significant changes in the oral microbial composition were observed. All strains and their respective LPS preparations induced periodontitis. Therefore, the ability to alter the lipid A composition in response to environmental conditions by lipid A phosphatases is required for both colonization of the rabbit and increases in the microbial load. Furthermore, the data demonstrate that multiple dysbiotic oral microbial communities can elicit periodontitis.

Topics: Animals; Bacterial Load; Bacteroidaceae Infections; Biota; Disease Models, Animal; Dysbiosis; Lipid A; Male; Periodontitis; Phosphoric Monoester Hydrolases; Porphyromonas gingivalis; Rabbits

The ectodomain of matrix protein 2 (M2e) of influenza virus is suggested to be a rational target for a universal influenza A vaccine. However, there are some concerns that M2e vaccines might not be highly effective in the general population with diverse genetic backgrounds. Here we examined the immunogenicity and protective efficacy of the baculovirus-derived virus-like particles containing multiple M2e (M2eVLP) with AS04 adjuvant in a C57BL/6 mouse strain (H-2(b)). M2eVLP vaccine induced significant levels of M2e-specific IgG in C57BL/6 mice after vaccination. Furthermore, M2eVLP adjuvanted with AS04 was more effective than M2eVLP alone in conferring protection as well as in inducing recall humoral and T cell responses specific for M2e after lethal influenza virus challenge. A mechanistic study provides evidence that activation of dendritic cells by the toll-like receptor 4 agonist MPL in the AS04 adjuvant was associated with interferon-γ producing CD4 T cell responses. Our results suggest that AS04 adjuvanted M2eVLP vaccines have the potential to improve cross-protection.

Topics: Adjuvants, Immunologic; Aluminum Hydroxide; Animals; Antibodies, Viral; CD4-Positive T-Lymphocytes; Disease Models, Animal; Female; Influenza Vaccines; Interferon-gamma; Lipid A; Mice, Inbred C57BL; Orthomyxoviridae Infections; Vaccines, Virus-Like Particle; Viral Matrix Proteins

The antioxidant N-acetyl-l-cysteine (NAC) had been shown to inhibit replication of seasonal human influenza A viruses. Here, the effects of NAC on H9N2 swine influenza virus-induced acute lung injury (ALI) were investigated in mice. BALB/c mice were inoculated intranasally with 10(7) 50% tissue culture infective doses (TCID(50)) of A/swine/HeBei/012/2008/(H9N2) viruses with or without NAC treatments to induce ALI model. The result showed that pulmonary inflammation, pulmonary edema, MPO activity, total cells, neutrophils, macrophages, TNF-α, IL-6, IL-1β and CXCL-10 in BALF were attenuated by NAC. Moreover, our data showed that NAC significantly inhibited the levels of TLR4 protein and TLR4 mRNA in the lungs. Pharmacological inhibitors of TLR4 (E5564) exerted similar effects like those determined for NAC in H9N2 swine influenza virus-infected mice. These results suggest that antioxidants like NAC represent a potential additional treatment option that could be considered in the case of an influenza A virus pandemic.

Topics: Acetylcysteine; Acute Lung Injury; Animals; Antioxidants; Cytokines; Disease Models, Animal; Humans; Inflammation Mediators; Influenza A Virus, H9N2 Subtype; Influenza, Human; Lipid A; Lung; Male; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Peroxidase; Swine; Toll-Like Receptor 4; Virus Replication

Several successful pathogens have evolved mechanisms to evade host defense, resulting in the establishment of persistent and chronic infections. One such pathogen, Porphyromonas gingivalis, induces chronic low-grade inflammation associated with local inflammatory bone loss and systemic inflammation manifested as atherosclerosis. P. gingivalis expresses an atypical lipopolysaccharide (LPS) structure containing heterogeneous lipid A species, that exhibit Toll-like receptor-4 (TLR4) agonist or antagonist activity, or are non-activating at TLR4. In this study, we utilized a series of P. gingivalis lipid A mutants to demonstrate that antagonistic lipid A structures enable the pathogen to evade TLR4-mediated bactericidal activity in macrophages resulting in systemic inflammation. Production of antagonistic lipid A was associated with the induction of low levels of TLR4-dependent proinflammatory mediators, failed activation of the inflammasome and increased bacterial survival in macrophages. Oral infection of ApoE(-/-) mice with the P. gingivalis strain expressing antagonistic lipid A resulted in vascular inflammation, macrophage accumulation and atherosclerosis progression. In contrast, a P. gingivalis strain producing exclusively agonistic lipid A augmented levels of proinflammatory mediators and activated the inflammasome in a caspase-11-dependent manner, resulting in host cell lysis and decreased bacterial survival. ApoE(-/-) mice infected with this strain exhibited diminished vascular inflammation, macrophage accumulation, and atherosclerosis progression. Notably, the ability of P. gingivalis to induce local inflammatory bone loss was independent of lipid A expression, indicative of distinct mechanisms for induction of local versus systemic inflammation by this pathogen. Collectively, our results point to a pivotal role for activation of the non-canonical inflammasome in P. gingivalis infection and demonstrate that P. gingivalis evades immune detection at TLR4 facilitating chronic inflammation in the vasculature. These studies support the emerging concept that pathogen-mediated chronic inflammatory disorders result from specific pathogen-mediated evasion strategies resulting in low-grade chronic inflammation.

Topics: Animals; Apolipoproteins E; Atherosclerosis; Bacteroidaceae Infections; Disease Models, Animal; HEK293 Cells; Humans; Lipid A; Macrophages; Mice; Osteoporosis; Porphyromonas gingivalis; Toll-Like Receptor 4; Vasculitis

Bacterial biofilm communities are associated with profound physiological changes that lead to novel properties compared to the properties of individual (planktonic) bacteria. The study of biofilm-associated phenotypes is an essential step toward control of deleterious effects of pathogenic biofilms. Here we investigated lipopolysaccharide (LPS) structural modifications in Escherichia coli biofilm bacteria, and we showed that all tested commensal and pathogenic E. coli biofilm bacteria display LPS modifications corresponding to an increased level of incorporation of palmitate acyl chain (palmitoylation) into lipid A compared to planktonic bacteria. Genetic analysis showed that lipid A palmitoylation in biofilms is mediated by the PagP enzyme, which is regulated by the histone-like protein repressor H-NS and the SlyA regulator. While lipid A palmitoylation does not influence bacterial adhesion, it weakens inflammatory response and enhances resistance to some antimicrobial peptides. Moreover, we showed that lipid A palmitoylation increases in vivo survival of biofilm bacteria in a clinically relevant model of catheter infection, potentially contributing to biofilm tolerance to host immune defenses. The widespread occurrence of increased lipid A palmitoylation in biofilms formed by all tested bacteria suggests that it constitutes a new biofilm-associated phenotype in Gram-negative bacteria.. Bacterial communities called biofilms display characteristic properties compared to isolated (planktonic) bacteria, suggesting that some molecules could be more particularly produced under biofilm conditions. We investigated biofilm-associated modifications occurring in the lipopolysaccharide (LPS), a major component of all Gram-negative bacterial outer membrane. We showed that all tested commensal and pathogenic biofilm bacteria display high incorporation of a palmitate acyl chain into the lipid A part of LPS. This lipid A palmitoylation is mediated by the PagP enzyme, whose expression in biofilm is controlled by the regulatory proteins H-NS and SlyA. We also showed that lipid A palmitoylation in biofilm bacteria reduces host inflammatory response and enhances their survival in an animal model of biofilm infections. While these results provide new insights into the biofilm lifestyle, they also suggest that the level of lipid A palmitoylation could be used as an indicator to monitor the development of biofilm infections on medical surfaces.

Topics: Acyltransferases; Animals; Bacterial Proteins; Biofilms; Catheter-Related Infections; Disease Models, Animal; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Gram-Negative Bacteria; Lipid A; Lipopolysaccharides; Lipoylation; Microbial Viability; Mutation; Phenotype; Rats; Transcription Factors

With the paucity of new drugs and HIV co-infection, vaccination remains an unmet research priority to combat visceral leishmaniasis (VL) requiring strong cellular immunity. Protein vaccination often suffers from low immunogenicity and poor generation of memory T cells for long-lasting protection. Cysteine proteases (CPs) are immunogenic proteins and key mediators of cellular functions in Leishmania. Here, we evaluated the vaccine efficacies of CPs against VL, using cationic liposomes with Toll like receptor agonists for stimulating host immunity against L. donovani in a hamster model.. Recombinant CPs type I (cpb), II (cpa) and III (cpc) of L. donovani were tested singly and in combination as a triple antigen cocktail for antileishmanial vaccination in hamsters. We found the antigens to be highly immunoreactive and persistent anti-CPA, anti-CPB and anti-CPC antibodies were detected in VL patients even after cure. The liposome-entrapped CPs with monophosphoryl lipid A-Trehalose dicorynomycolate (MPL-TDM) induced significantly high nitric oxide (up to 4 fold higher than controls) mediated antileishmanial activity in vitro, and resulted in strong in vivo protection. Among the three CPs, CPC emerged as the most potent vaccine candidate in combating the disease. Interestingly, a synergistic increase in protection was observed with liposomal CPA, CPB and CPC antigenic cocktail which reduced the organ parasite burden by 1013-1016 folds, and increased the disease-free survival of >80% animals at least up to 6 months post infection. Robust secretion of IFN-γ and IL-12, along with concomitant downregulation of Th2 cytokines, was observed in cocktail vaccinates, even after 3 months post infection.. The present study is the first report of a comparative efficacy of leishmanial CPs and their cocktail using liposomal formulation with MPL-TDM against L. donovani. The level of protection attained has not been reported for any other subcutaneous single or polyprotein vaccination against VL.

Topics: Animals; Antigens, Protozoan; Cations; Cord Factors; Cricetinae; Cysteine Proteases; Disease Models, Animal; Drug Delivery Systems; Leishmania donovani; Leishmaniasis; Lipid A; Liposomes; Mesocricetus; Protozoan Vaccines

Dendritic cells (DC) process and present antigens to T lymphocytes, inducing potent immune responses when encountered in association with activating signals, such as pathogen-associated molecular patterns. Using the 4T1 murine model of breast cancer, cationic liposomes containing monophosphoryl lipid A (MPL) and interleukin (IL)-12 were administered by intratumoral injection. Combination multivalent presentation of the Toll-like receptor-4 ligand MPL and cytotoxic 1,2-dioleoyl-3-trmethylammonium-propane lipids induced cell death, decreased cellular proliferation, and increased serum levels of IL-1β and tumor necrosis factor (TNF)-α. The addition of recombinant IL-12 further suppressed tumor growth and increased expression of IL-1β, TNF-α, and interferon-γ. IL-12 also increased the percentage of cytolytic T cells, DC, and F4/80(+) macrophages in the tumor. While single agent therapy elevated levels of nitric oxide synthase 3-fold above basal levels in the tumor, combination therapy with MPL cationic liposomes and IL-12 stimulated a 7-fold increase, supporting the observed cell cycle arrest (loss of Ki-67 expression) and apoptosis (TUNEL positive). In mice bearing dual tumors, the growth of distal, untreated tumors mirrored that of liposome-treated tumors, supporting the presence of a systemic immune response.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Immunohistochemistry; Interferon-gamma; Interleukin-12; Interleukin-1beta; Lipid A; Liposomes; Mice; Mice, Inbred BALB C; Tumor Necrosis Factor-alpha

Preconditioning with low doses of monophosphoryl lipid A (MPL) has been shown to induce endotoxin tolerance and to reduce the metabolic and hemodynamic consequences of endotoxin shock. However, no data are available about the effects of endotoxin preconditioning on cerebral metabolism during endotoxemia. The study was designed to determine the effects of endotoxin preconditioning with MPL on cerebral metabolism via microdialysis compared to muscle tissue metabolism during experimental endotoxemia.. In a controlled animal study, continuous endotoxin infusion (1μg/kg b.w. per h) was administrated to 7 female mixed-breed pigs after pretreatment with MPL in incremental doses of endotoxin during days 5-2 before the experiments. In the control group, 7 animals received a saline pretreatment. In addition to hemodynamic monitoring and blood gas analyses, interstitial lactate, pyruvate, glucose and glycerol concentrations in muscle and cerebral tissue were measured using in vivo microdialysis.. There were no significant differences between the two groups with respect to hemodynamic parameters, while mixed venous oxygen saturation (SvO2), arterial blood pH and mean pulmonary arterial pressure (MPAP) were significantly higher in the preconditioned group. Cerebral perfusion pressure (CPP) and brain tissue oxygen pressure (ptiO2) values stayed stable throughout the experiment with no inter-group differences. While interstitial concentrations of lactate and glycerol as well as the lactate/pyruvate (LP) and the lactate/glucose (LG) ratio in muscle tissues were significantly increased in control animals compared to those who had been pretreated with MPL; the results of cerebral microdialysis showed no significant changes in interstitial lactate or glycerol levels in both groups. However, the lactate/glucose (LG) ratio in the control group showed a significantly higher increase than in the preconditioned group.. Preconditioning with low doses of MPL ameliorates the negative metabolic effects of endotoxin shock in muscle tissue. With regard to cerebral metabolism, the present study suggests that MLP preconditioning provides moderate advantages, at least in an experimental model of endotoxin shock.

Topics: Animals; Cerebrum; Disease Models, Animal; Endotoxemia; Endotoxins; Female; Lipid A; Microdialysis; Shock, Septic; Swine

Topics: Animals; Atherosclerosis; Bacteroidaceae Infections; Disease Models, Animal; Immune Evasion; Lipid A; Mice; Periodontitis; Porphyromonas gingivalis

Non-replicating respiratory syncytial virus (RSV) vaccine candidates could potentially prime for enhanced respiratory disease (ERD) due to a T-cell-mediated immunopathology, following RSV infection. Vaccines with built-in immune response modifiers, such as Toll-like receptor (TLR) ligands, may avoid such aberrant imprinting of the immune system.. We developed reconstituted RSV envelopes (virosomes) with incorporated TLR4 ligand, monophosphoryl lipid A (RSV-MPLA virosomes). Immune responses and lung pathology after vaccination and challenge were investigated in ERD-prone cotton rats and compared with responses induced by live virus and formaldehyde-inactivated vaccine (FI-RSV), a known cause of ERD upon RSV challenge.. Vaccination with RSV-MPLA virosomes induced higher levels of virus-neutralizing antibodies than FI-RSV or live virus infection and provided protection against infection. FI-RSV, but not RSV-MPLA virosomes, primed for increases in expression of Th2 cytokines IL-4, IL-5, IL-13, and Th1 cytokine IL-1b, 6 hour-5 days after infection. By contrast, RSV-MPLA virosomes induced IFN-γ transcripts to similar levels as induced by live virus. Animals vaccinated with FI-RSV, but not RSV-MPLA virosomes showed alveolitis, with prominent neutrophil influx and peribronchiolar and perivascular infiltrates.. These results show that RSV-MPLA virosomes represent a safe and immunogenic vaccine candidate that warrants evaluation in a clinical setting.

Topics: Adjuvants, Immunologic; Animals; Disease Models, Animal; Female; Lipid A; Lung; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus Vaccines; Respiratory Syncytial Viruses; Sigmodontinae; Vaccines, Virosome

Gram-negative bacteria including Escherichia coli, Citrobacter rodentium, Salmonella typhimurium, and Shigella flexneri are sensed in an ill-defined manner by an intracellular inflammasome complex that activates caspase-11. We show that macrophages loaded with synthetic lipid A, E. coli lipopolysaccharide (LPS), or S. typhimurium LPS activate caspase-11 independently of the LPS receptor Toll-like receptor 4 (TLR4). Consistent with lipid A triggering the noncanonical inflammasome, LPS containing a divergent lipid A structure antagonized caspase-11 activation in response to E. coli LPS or Gram-negative bacteria. Moreover, LPS-mutant E. coli failed to activate caspase-11. Tlr4(-/-) mice primed with TLR3 agonist polyinosinic:polycytidylic acid [poly(I:C)] to induce pro-caspase-11 expression were as susceptible as wild-type mice were to sepsis induced by E. coli LPS. These data unveil a TLR4-independent mechanism for innate immune recognition of LPS.

Topics: Animals; Caspases; Caspases, Initiator; Cholera Toxin; Disease Models, Animal; Escherichia coli; Escherichia coli Infections; Immunity, Innate; Inflammasomes; Lipid A; Macrophages; Mice; Mice, Mutant Strains; Mutation; Salmonella Infections; Salmonella typhimurium; Sepsis; Toll-Like Receptor 4

Phosphoethanolamine (PEA) on Neisseria gonorrhoeae lipid A influences gonococcal inflammatory signaling and susceptibility to innate host defenses in in vitro models. Here, we evaluated the role of PEA-decorated gonococcal lipid A in competitive infections in female mice and in male volunteers. We inoculated mice and men with mixtures of wild-type N. gonorrhoeae and an isogenic mutant that lacks the PEA transferase, LptA. LptA production conferred a marked survival advantage for wild-type gonococci in the murine female genital tract and in the human male urethra. Our studies translate results from test tube to animal model and into the human host and demonstrate the utility of the mouse model for studies of virulence factors of the human-specific pathogen N. gonorrhoeae that interact with non-host-restricted elements of innate immunity. These results validate the use of gonococcal LptA as a potential target for development of novel immunoprophylactic strategies or antimicrobial treatments.. Gonorrhea is one of the most common bacterial sexually transmitted infections, and increasing antibiotic resistance threatens the use of currently available antimicrobial therapies. In this work, encompassing in vitro studies and in vivo studies of animal and human models of experimental genital tract infection, we document the importance of lipid A's structure, mediated by a single bacterial enzyme, LptA, in enhancing the fitness of Neisseria gonorrhoeae. The results of these studies suggest that novel agents targeting LptA may offer urgently needed prevention or treatment strategies for gonorrhea.

Topics: Animals; Disease Models, Animal; Ethanolaminephosphotransferase; Ethanolamines; Female; Gene Knockout Techniques; Gonorrhea; Healthy Volunteers; Humans; Lipid A; Male; Mice; Microbial Viability; Neisseria gonorrhoeae; Virulence; Virulence Factors

One potential strategy for the prevention of HIV infection is to induce virus specific mucosal antibody that can act as an immune barrier to prevent transmission. The mucosal application of chemokines after immunisation, termed "prime-pull", has been shown to recruit T cells to mucosal sites. We wished to determine whether this strategy could be used to increase B cells and antibody in the vaginal mucosa following immunisation with an HIV antigen. BALB/c mice were immunised intranasally with trimeric gp140 prior to vaginal application of the chemokine CCL28 or the synthetic TLR4 ligand MPLA, without antigen six days later. There was no increase in vaginal IgA, IgG or B cells following the application of CCL28, however vaginal application of MPLA led to a significant boost in antigen specific vaginal IgA. Follow up studies to investigate the effect of the timing of the "pull" stimulation demonstrated that when given 14 days after the initial immunisation MPLA significantly increased systemic antibody responses. We speculate that this may be due to residual inflammation prior to re-immunisation. Overall we conclude that in contrast to the previously observed effect on T cells, the use of "prime-pull" has only a modest effect on B cells and antibody.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Administration, Intravaginal; Animals; Antibody Specificity; Antigens, Viral; Disease Models, Animal; env Gene Products, Human Immunodeficiency Virus; Female; HIV Antibodies; HIV Infections; HIV-1; Immunization, Secondary; Immunoglobulin A; Lipid A; Mice

Francisella tularensis causes the zoonotic disease tularemia. Arthropod vectors are important transmission routes for the disease, although it is not known how Francisella survives the efficient arthropod immune response. Here, we used Drosophila melanogaster as a model host for Francisella infections and investigated whether the bacteria are resistant to insect humoral immune responses, in particular to the antimicrobial peptides (AMPs) secreted into the insect hemolymph. Moreover, we asked to what extent such resistance might depend on lipopolysaccharide (LPS) structure and surface characteristics of the bacteria. We analyzed Francisella novicida mutant strains in genes, directly or indirectly involved in specific steps of LPS biosynthesis, for virulence in wild-type and Relish(E20) immune-deficient flies, and tested selected mutants for sensitivity to AMPs in vitro. We demonstrate that Francisella is sensitive to specific fly AMPs, i.e. Attacin, Cecropin, Drosocin and Drosomycin. Furthermore, six bacterial genes, kpsF, manB, lpxF, slt, tolA and pal, were found to be required for resistance to Relish-dependent immune responses, illustrating the importance of structural details of Francisella lipid A and Kdo core for interactions with AMPs. Interestingly, a more negative surface charge and lack of O-antigen did not render mutant bacteria more sensitive to cationic AMPs and did not attenuate virulence in flies.

Topics: Animals; Antimicrobial Cationic Peptides; Arthropod Vectors; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Francisella tularensis; Genes, Bacterial; Immunity; Insect Proteins; Lipid A; Lipopolysaccharides; Mutation; Organisms, Genetically Modified; Sugar Acids; Transcription Factors; Tularemia

Synthesis of Escherichia coli LpxL, which transfers a secondary laurate chain to the 2' position of lipid A, in Yersinia pestis produced bisphosphoryl hexa-acylated lipid A at 37°C, leading to significant attenuation of virulence. Our previous observations also indicated that strain χ10015(pCD1Ap) (ΔlpxP32::P(lpxL) lpxL) stimulated a strong inflammatory reaction but sickened mice before recovery and retained virulence via intranasal (i.n.) infection. The development of live, attenuated Y. pestis vaccines may be facilitated by detoxification of its lipopolysaccharide (LPS). Heterologous expression of the lipid A 1-phosphatase, LpxE, from Francisella tularensis in Y. pestis yields predominantly 1-dephosphorylated lipid A, as confirmed by mass spectrometry. Results indicated that expression of LpxE on top of LpxL provided no significant reduction in virulence of Y. pestis in mice when it was administered i.n. but actually reduced the 50% lethal dose (LD(50)) by 3 orders of magnitude when the strain was administered subcutaneously (s.c.). Additionally, LpxE synthesis in wild-type Y. pestis KIM6+(pCD1Ap) led to slight attenuation by s.c. inoculation but no virulence change by i.n. inoculation in mice. In contrast to Salmonella enterica, expression of LpxE does not attenuate the virulence of Y. pestis.

Topics: Acyltransferases; Animals; Disease Models, Animal; Escherichia coli Proteins; Gene Expression; Lethal Dose 50; Lipid A; Mass Spectrometry; Membrane Proteins; Mice; Phosphoric Monoester Hydrolases; Plague; Recombinant Proteins; Survival Analysis; Virulence; Virulence Factors; Yersinia pestis

Lipopolysaccharide (LPS) structural modifications have been shown to specifically affect the pathogenesis of many gram-negative pathogens. In Francisella, modification of the lipid A component of LPS resulted in a molecule with no to low endotoxic activity. The role of the terminal lipid A phosphates in host recognition and pathogenesis was determined using a Francisella novicida mutant that lacked the 4' phosphatase enzyme (LpxF). The lipid A of this strain retained the phosphate moiety at the 4' position and the N-linked fatty acid at the 3' position on the diglucosamine backbone. Studies were undertaken to determine the pathogenesis of this mutant strain via the pulmonary and subcutaneous routes of infection. Mice infected with the lpxF-null F. novicida mutant by either route survived primary infection and subsequently developed protective immunity against a lethal wild-type (WT) F. novicida challenge. To determine the mechanism(s) by which the host controlled primary infection by the lpxF-null mutant, the role of innate immune components, including Toll-like receptor 2 (TLR2), TLR4, caspase-1, MyD88, alpha interferon (IFN-α), and gamma interferon(IFN-γ), was examined using knockout mice. Interestingly, only the IFN-γ knockout mice succumbed to a primary lpxF-null F. novicida mutant infection, highlighting the importance of IFN-γ production. To determine the role of components of the host adaptive immune system that elicit the long-term protective immune response, T- and B-cell deficient RAG1(-/-) mice were examined. All mice survived primary infection; however, RAG1(-/-) mice did not survive WT challenge, highlighting a role for T and B cells in the protective immune response.

Topics: Animals; Cytokines; Disease Models, Animal; Female; Francisella; Gene Knockout Techniques; Gram-Negative Bacterial Infections; Immunity, Innate; Lipid A; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphates; Phosphoric Monoester Hydrolases; Receptors, Immunologic; Survival Analysis; Virulence

Antimicrobial peptides (APs) belong to the arsenal of weapons of the innate immune system against infections. In the case of gram-negative bacteria, APs interact with the anionic lipid A moiety of the lipopolysaccharide (LPS). In yersiniae most virulence factors are temperature regulated. Studies from our laboratory demonstrated that Yersinia enterocolitica is more susceptible to polymyxin B, a model AP, when grown at 37°C than at 22°C (J. A. Bengoechea, R. Díaz, and I. Moriyón, Infect. Immun. 64:4891-4899, 1996), and here we have extended this observation to other APs, not structurally related to polymyxin B. Mechanistically, we demonstrate that the lipid A modifications with aminoarabinose and palmitate are downregulated at 37°C and that they contribute to AP resistance together with the LPS O-polysaccharide. Bacterial loads of lipid A mutants in Peyer's patches, liver, and spleen of orogastrically infected mice were lower than those of the wild-type strain at 3 and 7 days postinfection. PhoPQ and PmrAB two-component systems govern the expression of the loci required to modify lipid A with aminoarabinose and palmitate, and their expressions are also temperature regulated. Our findings support the notion that the temperature-dependent regulation of loci controlling lipid A modifications could be explained by H-NS-dependent negative regulation alleviated by RovA. In turn, our data also demonstrate that PhoPQ and PmrAB regulate positively the expression of rovA, the effect of PhoPQ being more important. However, rovA expression reached wild-type levels in the phoPQ pmrAB mutant background, hence indicating the existence of an unknown regulatory network controlling rovA expression in this background.

Topics: Animals; Antimicrobial Cationic Peptides; Arabinose; Bacterial Load; Bacterial Proteins; Disease Models, Animal; DNA-Binding Proteins; Drug Resistance, Bacterial; Gene Expression Regulation, Bacterial; Lipid A; Liver; Mice; O Antigens; Palmitates; Peyer's Patches; Polymyxin B; Spleen; Temperature; Transcription Factors; Yersinia enterocolitica; Yersinia Infections

Lipid A is a key component of the outer membrane of Gram-negative bacteria and stimulates proinflammatory responses via the Toll-like receptor 4 (TLR4)-MD2-CD14 pathway. Its endotoxic activity depends on the number and length of acyl chains and its phosphorylation state. In Salmonella enterica serovar Typhimurium, removal of the secondary laurate or myristate chain in lipid A results in bacterial attenuation and growth defects in vitro. However, the roles of the two lipid A phosphate groups in bacterial virulence and immunogenicity remain unknown. Here, we used an S. Typhimurium msbB pagL pagP lpxR mutant, carrying penta-acylated lipid A, as the parent strain to construct a series of mutants synthesizing 1-dephosphorylated, 4'-dephosphorylated, or nonphosphorylated penta-acylated lipid A. Dephosphorylated mutants exhibited increased sensitivity to deoxycholate and showed increased resistance to polymyxin B. Removal of both phosphate groups severely attenuated the mutants when administered orally to BALB/c mice, but the mutants colonized the lymphatic tissues and were sufficiently immunogenic to protect the host from challenge with wild-type S. Typhimurium. Mice receiving S. Typhimurium with 1-dephosphorylated or nonphosphorylated penta-acylated lipid A exhibited reduced levels of cytokines. Attenuated and dephosphorylated Salmonella vaccines were able to induce adaptive immunity against heterologous (PspA of Streptococcus pneumoniae) and homologous antigens (lipopolysaccharide [LPS] and outer membrane proteins [OMPs]).

Topics: Adaptive Immunity; Animals; Disease Models, Animal; Female; Humans; Immunity, Innate; Lipid A; Mice; Mice, Inbred BALB C; Phosphates; Salmonella Infections; Salmonella typhimurium; Salmonella Vaccines; Streptococcus pneumoniae; Vaccines, Attenuated; Virulence; Virulence Factors

Herpes Simplex Virus Type 2 (HSV-2) infection can result in life-long recurrent genital disease, asymptomatic virus shedding, and transmission. No vaccine to date has shown significant protection clinically. Here, we used a mouse model of genital HSV-2 infection to test the efficacy of a vaccine consisting of whole, formalin-inactivated HSV-2 (FI-HSV2) formulated with monophosphoryl lipid A (MPL) and alum adjuvants. Vaccine components were administered alone or as a prime-boost immunization together with DNA vaccines encoding a truncated glycoprotein D2 (gD2t) and two conserved HSV-2 genes necessary for virus replication, UL5 (DNA helicase) and UL30 (DNA polymerase). Our results show: (1) compared with mock immunized controls, mice immunized with FI-HSV2 plus MPL/alum consistently showed protection against disease burden and total viral shedding while the mice immunized with gD2t protein with MPL/alum did not; (2) protection against genital disease and viral replication correlated with the type of boost in a prime-boost immunization with little advantage afforded by a DNA prime; (3) intramuscular (i.m.) immunization with FI-HSV2 in MPL/Alhydrogel adjuvant provided nearly complete protection against vaginal HSV-2 shedding after a lethal intravaginal (i.vag.) short-term challenge and long-term rechallenge; (4) single formulation immunization with DNA vaccines, FI-HSV2, and MPL in an aluminum phosphate (Adju-Phos) adjuvant did not increase protection relative to FI-HSV2/MPL/Adju-Phos alone; and (5) addition of MPL/alum to the FI-HSV2 was required for optimal protection against disease, viral replication, and latent virus load in the dorsal root ganglia (DRG). Most notably, an optimized vaccine formulation of FI-HSV2 MPL/Alhydrogel given i.m. completely protected against detectable vaginal HSV-2 shedding in the majority of animals and HSV-2 latent DNA in the DRG of all animals.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antigens, Viral; Disease Models, Animal; Female; Ganglia, Spinal; Herpes Genitalis; Herpesvirus 2, Human; Herpesvirus Vaccines; Lipid A; Mice; Mice, Inbred BALB C; Survival Analysis; Vaccination; Vaccines, DNA; Vagina; Virus Shedding

Numerous experimental Leishmania vaccines have been developed to prevent the visceral and cutaneous forms of Leishmaniasis, which occur after exposure to the bite of an infected sand fly, yet only one is under evaluation in humans. KSAC and L110f, recombinant Leishmania polyproteins delivered in a stable emulsion (SE) with the TLR4 agonists monophosphoryl lipid A or glucopyranosyl lipid A (GLA) have shown protection in animal models. KSAC+GLA-SE protected against cutaneous disease following sand fly transmission of Leishmania major in susceptible BALB/c mice. Similar polyprotein adjuvant combinations are the vaccine candidates most likely to see clinical evaluation. We assessed immunity generated by KSAC or L110f vaccination with GLA-SE following challenge with L. major by needle or infected sand fly bite in resistant C57BL/6 mice. Polyprotein-vaccinated mice had a 60-fold increase in CD4(+)IFN-γ(+) T cell numbers versus control animals at 2 wk post-needle inoculation of L. major, and this correlated with a 100-fold reduction in parasite load. Immunity did not, however, reach levels observed in mice with a healed primary infection. Following challenge by infected sand fly bite, polyprotein-vaccinated animals had comparable parasite loads, greater numbers of neutrophils at the challenge site, and reduced CD4(+)IFN-γ(+)/IL-17(+) ratios versus nonvaccinated controls. In contrast, healed animals had significantly reduced parasite loads and higher CD4(+)IFN-γ(+)/IL-17(+) ratios. These observations demonstrate that vaccine-induced protection against needle challenge does not necessarily translate to protection following challenge by infected sand fly bite.

Topics: Adjuvants, Immunologic; Animals; CD4-Positive T-Lymphocytes; Disease Models, Animal; Emulsions; Interferon-gamma; Interleukin-17; Leishmania major; Leishmaniasis Vaccines; Leishmaniasis, Cutaneous; Lipid A; Mice; Mice, Inbred BALB C; Protozoan Proteins; Psychodidae; Recombinant Proteins

Visceral leishmaniasis, a chronic systemic infection, is the major cause of morbidity and mortality in many parts of world. The current drugs for the treatment of leishmaniasis are toxic, expensive, difficult to administer and becoming ineffective due to the emergence of drug resistance. In the absence of effective treatment, vaccination remains the only hope for control of the disease. We have evaluated the protective efficacy of two heat shock proteins (Hsp70 and Hsp83) in combination with two different adjuvants (MPLA and ALD) in Leishmania donovani infected inbred BALB/c mice. The proteins were isolated by SDS-PAGE and the mice were immunized subcutaneously with Hsp70+Hsp83, Hsp70+Hsp83+ALD and Hsp70+Hsp83+MPLA. These were challenged with 10(7) promastigotes of L. donovani. The animals were sacrificed on 30, 60 and 90 days post challenge for the assessment of parasite load and generation of cellular and humoral immune responses. The vaccines induced a strong protective response against experimental visceral leishmaniasis as shown by reduced parasite load in liver of all immunized groups as compared to the infected controls. The vaccines also led to the augmentation of DTH responses, increased levels of IgG2a, IFN-γ and IL-2. Both the adjuvants raised significantly the level of protection imparted by the proteins but MPLA was more effective in comparison to ALD.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Protozoan; Antigens, Protozoan; Cytokines; Disease Models, Animal; HSP70 Heat-Shock Proteins; Hypersensitivity, Delayed; Immunity, Humoral; Leishmania donovani; Leishmaniasis Vaccines; Leishmaniasis, Visceral; Lipid A; Liver; Mice; Mice, Inbred BALB C

Despite several attempts to develop an effective prophylactic vaccine for HSV-2, all have failed to show efficacy in the clinic. The most recent of these failures was the GlaxoSmithKline (GSK) subunit vaccine based on the glycoprotein gD with the adjuvant monophosphoryl lipid A (MPL). In a phase 3 clinical trial, this vaccine failed to protect from HSV-2 disease, even though good neutralizing antibody responses were elicited. We aimed to develop a superior, novel HSV-2 vaccine containing either gD or gB alone or in combination, together with the potent adjuvant CpG oligodeoxynucleotides (CPG). The immunogenic properties of these vaccines were compared in mice. We show that gB/CPG/alum elicited a neutralizing antibody response similar to that elicited by gD/CPG/alum vaccine but a significantly greater gamma interferon (IFN-γ) T cell response. Furthermore, the combined gB-gD/CPG/alum vaccine elicited significantly greater neutralizing antibody and T cell responses than gD/MPL/alum. The efficacies of these candidate vaccines were compared in the mouse and guinea pig disease models, including a novel male guinea pig genital disease model. These studies demonstrated that increased immune response did not correlate to improved protection. First, despite a lower IFN-γ T cell response, the gD/CPG/alum vaccine was more effective than gB/CPG/alum in mice. Furthermore, the gB-gD/CPG/alum vaccine was no more effective than gD/MPL/alum in mice or male guinea pigs. We conclude that difficulties in correlating immune responses to efficacy in animal models will act as a deterrent to researchers attempting to develop effective HSV vaccines.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibodies, Neutralizing; Antibodies, Viral; Disease Models, Animal; Guinea Pigs; Herpes Genitalis; Herpesvirus 2, Human; Herpesvirus Vaccines; Interferon-gamma; Lipid A; Mice; Oligodeoxyribonucleotides; Severity of Illness Index; T-Lymphocytes; Viral Envelope Proteins

Diabetes mellitus and obesity, which confer an increased risk of sudden cardiac death, are associated with cardiomyocyte lipid accumulation and altered cardiac electric properties, manifested by prolongation of the QRS duration and QT interval. It is difficult to distinguish the contribution of cardiomyocyte lipid accumulation from the contribution of global metabolic defects to the increased incidence of sudden death and electric abnormalities.. In order to study the effects of metabolic abnormalities on arrhythmias without the complex systemic effects of diabetes mellitus and obesity, we studied transgenic mice with cardiac-specific overexpression of peroxisome proliferator-activated receptor γ 1 (PPARγ1) via the cardiac α-myosin heavy-chain promoter. The PPARγ transgenic mice develop abnormal accumulation of intracellular lipids and die as young adults before any significant reduction in systolic function. Using implantable ECG telemeters, we found that these mice have prolongation of the QRS and QT intervals and spontaneous ventricular arrhythmias, including polymorphic ventricular tachycardia and ventricular fibrillation. Isolated cardiomyocytes demonstrated prolonged action potential duration caused by reduced expression and function of the potassium channels responsible for repolarization. Short-term exposure to pioglitazone, a PPARγ agonist, had no effect on mortality or rhythm in WT mice but further exacerbated the arrhythmic phenotype and increased the mortality in the PPARγ transgenic mice.. Our findings support an important link between PPARγ activation, cardiomyocyte lipid accumulation, ion channel remodeling, and increased cardiac mortality.

Topics: Action Potentials; Animals; Calcium; Connexin 43; Death, Sudden, Cardiac; Disease Models, Animal; Electrocardiography; Hypoglycemic Agents; Incidence; Lipid A; Mice; Mice, Transgenic; Myocardium; Myocytes, Cardiac; Phenotype; Pioglitazone; Potassium; Potassium Channels, Voltage-Gated; PPAR gamma; Refractory Period, Electrophysiological; Sodium; Tachycardia, Ventricular; Thiazolidinediones; Ventricular Fibrillation; Ventricular Remodeling

Vaccines that activate strong specific Th1-predominant immune responses are critically needed for many intracellular pathogens, including Leishmania. The requirement for sustained and efficient vaccination against leishmaniasis is to formulate the best combination of immunopotentiating adjuvant with the stable antigen (Ag) delivery system. The aim of the present study is to evaluate the effectiveness of an immunomodulator on liposomal Ag through subcutaneous (s.c.) route of immunization, and its usefulness during prime/boost against visceral leishmaniasis (VL) in BALB/c mice.. Towards this goal, we formulated recombinant GP63 (rGP63)-based vaccines either with monophosphoryl lipid A-trehalose dicorynomycolate (MPL-TDM) or entrapped within cationic liposomes or both. Combinatorial administration of liposomes with MPL-TDM during prime confers activation of dendritic cells, and induces an early robust T cell response. To investigate whether the combined formulation is required for optimum immune response during boost as well, we chose to evaluate the vaccine efficacy in mice primed with combined adjuvant system followed by boosting with either rGP63 alone, in association with MPL-TDM, liposomes or both. We provide evidences that the presence of either liposomal rGP63 or combined formulations during boost is necessary for effective Th1 immune responses (IFN-γ, IL-12, NO) before challenge infection. However, boosting with MPL-TDM in conjugation with liposomal rGP63 resulted in a greater number of IFN-γ producing effector T cells, significantly higher levels of splenocyte proliferation, and Th1 responses compared to mice boosted with liposomal rGP63, after virulent Leishmania donovani (L. donovani) challenge. Moreover, combined formulations offered superior protection against intracellular amastigote replication in macrophages in vitro, and hepatic and splenic parasite load in vivo.. Our results define the immunopotentiating effect of MPL-TDM on protein Ag encapsulated in a controlled release system against experimental VL.

Topics: Adjuvants, Immunologic; Animals; Cord Factors; Cytokines; Disease Models, Animal; Female; Humans; Injections, Subcutaneous; Leishmania donovani; Leishmaniasis Vaccines; Leishmaniasis, Visceral; Lipid A; Liposomes; Metalloendopeptidases; Mice; Mice, Inbred BALB C; Nitric Oxide; Phosphatidylcholines; Th1 Cells; Vaccination; Vaccines, Synthetic

Lipopolysaccharide (LPS), a major component of the meningococcal outer membrane, is sensed by the host through activation of Toll-like receptor 4 (TLR4). Recently, we demonstrated that a surprisingly large fraction of Neisseria meningitidis disease isolates are lipid A mutants, due to inactivating mutations in the lpxL1 gene. The lpxL1 mutants activate human TLR4 much less efficiently than wild-type bacteria, which may be advantageous by allowing them to escape from the innate immune system. Here we investigated the influence of lipid A structure on virulence in a mouse model of meningococcal sepsis. One limitation, however, is that murine TLR4 recognizes lpxL1 mutant bacteria much better than human TLR4. We show that an lpxL2 mutant, another lipid A mutant lacking an acyl chain at a different position, activates murine TLR4 less efficiently than the lpxL1 mutant. Therefore, the lpxL2 mutant in mice might be a better model for infections with lpxL1 mutants in humans. Interestingly, we found that the lpxL2 mutant is more virulent in mice than the wild-type strain, whereas the lpxL1 mutant is actually much less virulent than the wild-type strain. These results demonstrate the crucial role of N. meningitidis lipid A structure in virulence.

Topics: Animals; Bacteremia; Bacterial Outer Membrane Proteins; Cell Line; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Immunity, Innate; Lipid A; Meningococcal Infections; Mice; Mice, Inbred C57BL; Mutation; Neisseria meningitidis; Toll-Like Receptor 4

The transmembrane protein Toll-like receptor 4 (TLR4), which exists mainly in macrophages such as Kupffer cells of the liver, plays an important role in recognizing and mediating macrophage activation and pro-inflammatory cytokine release. Activation of the pro-inflammatory cytokine cascade, including tumor necrosis factor-alpha (TNF-alpha), has a pivotal role in the progression of severe liver injury. D-galactosamine (GalN) and lipopolysaccharide (LPS)-induced liver injury in rats is an experimental model of fulminant hepatic failure, where TNF-alpha plays a central role in the progression of liver injury. E5564, a synthetic analogue of the lipid A component of endotoxin, inhibits endotoxin-stimulated inflammation and is under study for patients with sepsis. In the present study, we sought to explore the salvage effect of TLR4 antagonist E5564 on GalN+LPS-induced acute liver failure (ALF) in rats.. ALF was induced in male Wistar rats by the intraperitoneal injection of GalN (500 mg/kg) and LPS (50 microg/kg). Immediately after GalN+LPS injection, rats were treated with intravenous injection of E5564 (3 mg/kg). The cumulative survival rates of GalN+LPS-induced ALF rats were compared between those with and without E5564 treatment.. The intravenous injection of E5564 reduced the elevation of serum total bilirubin, aspartate aminotransferase, alanine aminotransferase and TNF-alpha levels in rats at 3 h after GalN+LPS injection, and improved the survival rate of GalN+LPS-induced ALF rats at 24 h (8% vs 43%).. TLR4 antagonist E5564 reduced GalN+LPS-induced acute liver injury in rats and improved the overall survival rate of GalN+LPS-induced ALF rats. It may contribute to the treatment of ALF through blocking endotoxin-induced TNF-alpha overproduction of macrophages.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Bilirubin; Biomarkers; Disease Models, Animal; Galactosamine; Injections, Intravenous; Lipid A; Lipopolysaccharides; Liver; Liver Failure, Acute; Male; Rats; Rats, Wistar; Time Factors; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Infections are the most frequent cause of complications in trauma patients. Post-traumatic immune suppression (IS) exposes patients to pneumonia (PN). The main pathogen involved in PN is Methicillin Susceptible Staphylococcus aureus (MSSA). Dendritic cells () may be centrally involved in the IS. We assessed the consequences of hemorrhage on pneumonia outcomes and investigated its consequences on DCs functions. A murine model of hemorrhagic shock with a subsequent MSSA pneumonia was used. Hemorrhage decreased the survival rate of infected mice, increased systemic dissemination of sepsis and worsened inflammatory lung lesions. The mRNA expression of Tumor Necrosis Factor-alpha (TNF-α), Interferon-beta (IFN-β) and Interleukin (IL)-12p40 were mitigated for hemorrhaged-mice. The effects of hemorrhage on subsequent PN were apparent on the pDCs phenotype (reduced MHC class II, CD80, and CD86 molecule membrane expression). In addition, hemorrhage dramatically decreased CD8(+) cDCs- and CD8(-) cDCs-induced allogeneic T-cell proliferation during PN compared with mice that did not undergo hemorrhage. In conclusion, hemorrhage increased morbidity and mortality associated with PN; induced severe phenotypic disturbances of the pDCs subset and functional alterations of the cDCs subset. After hemorrhage, a preventive treatment with CpG-ODN or Monophosphoryl Lipid A increased transcriptional activity in DCs (TNF-α, IFN-β and IL-12p40) and decreased mortality of post-hemorrhage MSSA pneumonia.

Topics: Animals; Cell Proliferation; Cytokines; Disease Models, Animal; Lipid A; Mice; Oligodeoxyribonucleotides; Pilot Projects; Pneumonia, Bacterial; RNA, Messenger; Shock, Hemorrhagic; Staphylococcus aureus; T-Lymphocytes

Biliary atresia (BA) is a typical cholestatic neonatal disease, characterized by obliteration of intra- and/or extra-hepatic bile ducts. However, the mechanisms contributing to the pathogenesis of BA remain uncertain. Because of decreased bile flow, infectious complications and damaging endotoxemia occur frequently in patients with BA. The aim of this study was to investigate endotoxin levels in patients with BA and the relation of these levels with the expression of the endotoxin receptor, CD14.. The plasma levels of endotoxin and soluble CD14 were measured with a pyrochrome Limulus amebocyte lysate assay and enzyme-linked immunosorbent assay in patients with early-stage BA when they received the Kasai procedure (KP), in patients who were jaundice-free post-KP and followed-up at the outpatient department, in patients with late-stage BA when they received liver transplantation, and in patients with choledochal cysts. The correlation of CD14 expression with endotoxin levels in rats following common bile duct ligation was investigated.. The results demonstrated a significantly higher hepatic CD14 mRNA and soluble CD14 plasma levels in patients with early-stage BA relative to those with late-stage BA. However, plasma endotoxin levels were significantly higher in both the early and late stages of BA relative to controls. In rat model, the results demonstrated that both endotoxin and CD14 levels were significantly increased in liver tissues of rats following bile duct ligation.. The significant increase in plasma endotoxin and soluble CD14 levels during BA implies a possible involvement of endotoxin stimulated CD14 production by hepatocytes in the early stage of BA for removal of endotoxin; whereas, endotoxin signaling likely induced liver injury and impaired soluble CD14 synthesis in the late stages of BA.

Topics: Alanine Transaminase; Animals; Biliary Atresia; Bilirubin; Disease Models, Animal; Disease Progression; Endotoxins; Female; Gene Expression Regulation; Humans; Infant; Lipid A; Lipopolysaccharide Receptors; Liver; Male; Rats; Rats, Sprague-Dawley; RNA, Messenger; Solubility

The acquisition of immunity following subclinical or resolved infection with the intracellular parasite Leishmania donovani suggests that vaccination could prevent visceral leishmaniasis. The characteristics and in vitro stimulating capability of the recombinant proteins expressed by previously identified clones on the basis of their capacity to stimulate an indigenously established Leishmania-specific cell line leading to high level of IFN-gamma suggested these to be potential candidates for immunoprophylaxis against leishmaniasis. In this study, we investigated the protective efficacy of purified recombinant proteins from two of the identified cDNA clones along with the adjuvant MPL, in a hamster model of experimental leishmaniasis. We demonstrate here that the immunization of animals with one of the recombinant proteins (rF14) having 97% similarity to C1 clone of L. chagasi ribosomal protein gene P0 (rLiP0) along with MPL provided partial protection against the virulent challenge of L. donovani. The absence of antigen-specific lymphoproliferative responses in these immunized animals may be responsible for the lack of complete and long-lasting protection.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Protozoan; Antigens, Protozoan; Cricetinae; Cytokines; Disease Models, Animal; Female; Granuloma; Hypersensitivity, Delayed; Immunoglobulin G; Leishmania donovani; Leishmaniasis, Visceral; Lipid A; Liver; Lymphocyte Activation; Male; Mesocricetus; Nitrites; Organ Size; Protozoan Vaccines; Spleen; Superoxides; Vaccines, Synthetic

Toll-like receptor 4 (TLR4) is a transmembrane protein, existing mainly in macrophages, such as Kupffer cells of the liver. It plays an important role in recognizing and mediating macrophage activation and pro-inflammatory cytokine release. Activation of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-alpha is pivotal in the progression of liver injury. Gut-derived endotoxin has been considered to play an important role in the development and progression of a D-galactosamine (GalN)-induced acute severe liver injury (ALI) model. E5564, a synthetic analog of the lipid A component of endotoxin, inhibits endotoxin-stimulated inflammation and is under study for patients with sepsis. In this study, we seek to explore the effect of TLR4 antagonist E5564 on GalN-induced ALI in rats.. ALI was induced in male Wistar rats by the i.p. injection of 1 g/kg bodyweight of GalN and immediately after GalN injection they were treated with an i.v. injection of 3 mg/kg bodyweight of E5564. At 24 h after GalN injection with or without E5564, serum levels of total bilirubin (T.Bil), alanine aminotransferase (ALT) and TNF-alpha were analyzed. Expression levels of TNF-alpha, TLR4 and CD14 mRNA in the whole liver of rats was detected by reverse transcription polymerase chain reaction analysis.. The i.v. injection of E5564 reduced the elevation of serum T.Bil, ALT and TNF-alpha levels in rats treated with GalN. The expression level of TNF-alpha mRNA in the whole liver, which was increased at 24 h after GalN injection, was also reduced by i.v. injection of E5564.. TLR4 antagonist E5564 reduced GalN-induced ALI in rats. It may contribute to the treatment of acute liver failure through blocking endotoxin-induced TNF-alpha overproduction of macrophages.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Bilirubin; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Galactosamine; Lipid A; Liver Failure, Acute; Male; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

This study analysed the effect of priming the innate immune system using synthetic lipid A mimetics in a Yersinia pestis murine pulmonary infection model. Two aminoalkyl glucosaminide 4-phosphate (AGP) Toll-like receptor 4 (TLR4) ligands, delivered intranasally, extended time to death or protected against a lethal Y. pestis CO92 challenge. The level of protection was dependent upon the challenge dose of Y. pestis and the timing of AGP therapy. Protection correlated with cytokine induction and a decreased bacterial burden in lung tissue. AGP protection was TLR4-dependent and was not evidenced in transgenic TLR4-deficient mice. AGP therapy augmented with subtherapeutic doses of gentamicin produced dramatically enhanced survival. Combined, these results indicated that AGPs may be useful in protection of immunologically naive individuals against plague and potentially other infectious agents, and that AGP therapy may be used synergistically with other therapies.

Topics: Animals; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Glucosamine; Humans; Immunity, Innate; Lipid A; Lung; Mice; Mice, Inbred BALB C; Mice, Knockout; Organophosphorus Compounds; Plague; Specific Pathogen-Free Organisms; Time Factors; Toll-Like Receptor 4; Yersinia pestis

An effective intranasal (i.n.) vaccine against pneumonic plague was developed. The formulation employed two synthetic lipid A mimetics as adjuvant combined with Yersinia pestis-derived V- and F1-protective antigens. The two nontoxic lipid A mimetics, classed as amino-alkyl glucosaminide 4-phosphates (AGPs) are potent ligands for the Toll-like receptor (TLR) 4. Using a murine (BALB/c) pneumonic plague model, we showed a single i.n. application of the vaccine provided 63% protection within 21 days against a Y. pestis CO92 100 LD50 challenge. Protection reached 100% by 150 days. Using a homologous i.n. 1 degrees /2 degrees dose regimen, with the boost administered at varying times, 63% protection was achieved within 7 days and 100% protection was achieved by 21 days after the first immunization. Little or no protection was observed in animals that received antigens alone, and no protection was observed when the vaccine was administered to BALB/c TLR4 mutant mice. Vaccine-induced serum IgG titers to F1 and V-antigen were reflected in high titers for IgG1 and IgG2a, the latter reflecting a bias for a cell-mediated (TH1) immune response. This intranasal vaccine showed 90% protection in Sprague-Dawley rats challenged with 1000 LD50. We conclude that lipid A mimetics are highly effective adjuvants for an i.n. plague vaccine.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Antibodies, Bacterial; Disease Models, Animal; Female; Glucosamine; Humans; Lipid A; Male; Mice; Mice, Inbred BALB C; Molecular Mimicry; Plague; Plague Vaccine; Rats; Rats, Sprague-Dawley; Toll-Like Receptor 4; Yersinia pestis

Francisella tularensis (Ft) is a highly infectious gram-negative bacterium and the causative agent of the human disease tularemia. Ft is designated a class A select agent by the Centers for Disease Control and Prevention. Human clinical isolates of Ft produce lipid A of similar structure to Ft subspecies novicida (Fn), a pathogen of mice. We identified three enzymes required for Fn lipid A carbohydrate modifications, specifically the presence of mannose (flmF1), galactosamine (flmF2), or both carbohydrates (flmK). Mutants lacking either galactosamine (flmF2) or galactosamine/mannose (flmK) addition to their lipid A were attenuated in mice by both pulmonary and subcutaneous routes of infection. In addition, aerosolization of the mutants (flmF2 and flmK) provided protection against challenge with wild-type (WT) Fn, whereas subcutaneous administration of only the flmK mutant provided protection from challenge with WT Fn. Furthermore, infection of an alveolar macrophage cell line by the flmK mutant induced higher levels of tumor necrosis factor-alpha (TNF-alpha) and macrophage inhibitory protein-2 (MIP-2) when compared to infection with WT Fn. Bone marrow-derived macrophages (BMMø) from Toll-like receptor 4 (TLR4) and TLR2/4 knockout mice infected with the flmK mutant also produced significantly higher amounts of interleukin-6 (IL-6) and MIP-2 than BMMø infected with WT Fn. However, production of IL-6 and MIP-2 was undetectable in BMMø from MyD88(-/-) mice infected with either strain. MyD88(-/-) mice were also susceptible to flmK mutant infection. We hypothesize that the ability of the flmK mutant to activate pro-inflammatory cytokine/chemokine production and innate immune responses mediated by the MyD88 signaling pathway may be responsible for its attenuation, leading to the induction of protective immunity by this mutant.

Topics: Animals; Biomarkers; Bone Marrow Cells; Cell Line; Disease Models, Animal; Female; Francisella tularensis; Gene Silencing; Genes, Bacterial; Immunity, Innate; Lipid A; Macrophages, Alveolar; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mutation; Myeloid Differentiation Factor 88; Signal Transduction; Specific Pathogen-Free Organisms; Tularemia

Cystathionine beta-synthase-deficient mice (Cbs(-/-)) exhibit several pathophysiological features similar to hyperhomocysteinemic patients, including endothelial dysfunction and hepatic steatosis. Heterozygous mutants (Cbs(+/-)) on the C57BL/6J background are extensively analyzed in laboratories worldwide; however, detailed analyses of Cbs(-/-) have been hampered by the fact that they rarely survive past the weaning age probably due to severe hepatic dysfunction. We backcrossed the mutants with four inbred strains (C57BL/6J(Jcl), BALB/cA, C3H/HeJ and DBA/2J) for seven generations, and compared Cbs(-/-) phenotypes among the different genetic backgrounds. Although Cbs(-/-) on all backgrounds were hyperhomocysteinemic/hypermethioninemic and suffered from lipidosis/hepatic steatosis at 2 weeks of age, >30% of C3H/HeJ-Cbs(-/-) survived over 8 weeks whereas none of DBA/2J-Cbs(-/-) survived beyond 5 weeks. At 2 weeks, serum levels of total homocysteine and triglyceride were lowest in C3H/HeJ-Cbs(-/-). Adult C3H/HeJ-Cbs(-/-) survivors showed hyperhomocysteinemia but escaped hypermethioninemia, lipidosis and hepatic steatosis. They appeared normal in general behavioral tests but showed cerebellar malformation and impaired learning ability in the passive avoidance step-through test, and required sufficient dietary supplementation of cyst(e)ine for survival, demonstrating the essential roles of cystathionine beta-synthase in the central nervous system function and cysteine biosynthesis. Our C3H/HeJ-Cbs(-/-) mice could be useful tools for investigating clinical symptoms such as mental retardation and thromboembolism that are found in homocysteinemic patients.

Topics: Amino Acids; Animals; Behavior, Animal; Cerebellar Diseases; Cystathionine beta-Synthase; Cysteine; Disease Models, Animal; Female; Humans; Hyperhomocysteinemia; Kaplan-Meier Estimate; Lipid A; Male; Mice; Mice, Inbred Strains; Mice, Knockout; Phosphatidylcholine-Sterol O-Acyltransferase; Species Specificity

A DNA plasmid encoding human immunodeficiency virus type 1 (HIV-1) env, nef and tat genes was used in mice in a prime-boost immunization regimen with the corresponding recombinant proteins. The genetic immunogen was delivered with a gene gun and the proteins were injected intramuscularly together with the adjuvant AS02A. Immunizations were followed by experimental challenge with pseudotyped HIV-1 subtype A or B virus. In an initial experiment in which animals were challenged four weeks after the final immunization, all single modality and prime-boost vaccinations resulted in a significant level of protection as compared to control animals. There was a trend for DNA-alone immunization yielding the highest protection. In a subsequent study, a late challenge was performed 19 weeks after the final immunization. All groups having received the DNA vaccine, either alone or in combination with adjuvanted protein, exhibited strong protection against HIV replication. The subtype-specific protection against the experimental HIV challenge was significantly stronger than the cross-protection. Cellular and humoral immune responses were assessed during immunization and after challenge, but without clear correlation to protection against HIV replication. The data suggest that either DNA or protein antigens alone provide partial protection against an HIV-1/MuLV challenge and that DNA immunization is essential for achieving very high levels of efficacy in this murine HIV-1 challenge model. While prime-boost combinations were more immunogenic than DNA alone, they did not appear to provide any further enhancement over DNA vaccine mediated efficacy. The DNA immunogen might prime low levels of CD8+ T cells responsible for virus clearance or possibly a yet unidentified mechanism of protection.

Topics: Adjuvants, Immunologic; AIDS Vaccines; Animals; Biolistics; Disease Models, Animal; Drug Combinations; HIV Antibodies; HIV Antigens; HIV Infections; HIV-1; Humans; Immunization, Secondary; Injections, Intramuscular; Leukemia Virus, Murine; Lipid A; Mice; Mice, Inbred C57BL; Mice, Transgenic; Recombinant Proteins; Retroviridae Infections; Saponins; T-Lymphocytes; Tumor Virus Infections; Vaccination; Vaccines, DNA; Vaccines, Subunit

Sepsis and endotoxaemia are important causes of morbidity and mortality in humans. Research on sepsis focuses on rodent models most of which are poorly responsive to lipopolysaccharide (LPS), and thus do not mimic very well the high sensitivity of humans. Therefore, there is a need to develop more clinically relevant models. Horses suffer from a similar endotoxaemic syndrome to humans with high morbidity and mortality. LPS analogues that act as antagonists at Toll-like receptor 4 (TLR4) are being developed as novel treatments for endotoxaemia. Due to differences in recognition of ligands by TLR4 from different mammalian species, individual LPS molecules may act as agonists in some species and antagonists in others. The synthetic lipid A analogue E5531 is an antagonist at TLR4 in humans and mice, but its effects at TLR4 from other species are unknown. In the studies reported here, Escherichia coli LPS is a full agonist on equine bone marrow macrophage-like cells and its effects are antagonised by E5531. Similarly, E. coli LPS is an agonist and E5531 an antagonist on monocytes isolated from peripheral blood of healthy horses and human embryonic kidney (HEK) cells, transiently transfected to express horse TLR4 and its associated cell surface proteins MD2 and CD14. In contrast, both E. coli LPS and E5531 behave as agonists in horse whole blood by inducing production of equivalent amounts of the inflammatory mediator prostaglandin. This finding suggests that modification of E5531 may occur in whole blood, for example, deacylation, which alters its activity. This comparative study has revealed a novel pharmacological action of E5531 and emphasises the importance of extending studies of this nature beyond the normal rodent models.

Topics: Animals; Cell Line; Dinoprostone; Disease Models, Animal; Endotoxemia; Epoprostenol; Female; Humans; In Vitro Techniques; Interferon-gamma; Lipid A; Lipopolysaccharides; Male; Mice; Monocytes; Recombinant Proteins; Sepsis; Species Specificity; Toll-Like Receptor 4; Transfection

Klebsiella pneumoniae is an important cause of nosocomial Gram-negative sepsis. Lipopolysaccharide (LPS) is considered to be a major virulence determinant of this encapsulated bacterium and most mutations to the lipid A anchor of LPS are conditionally lethal to the bacterium. We studied the role of LPS acylation in K. pneumoniae disease pathogenesis by using a mutation of lpxM (msbB/waaN), which encodes the enzyme responsible for late secondary acylation of immature lipid A molecules. A K. pneumoniae B5055 (K2:O1) lpxM mutant was found to be attenuated for growth in the lungs in a mouse pneumonia model leading to reduced lethality of the bacterium. B5055DeltalpxM exhibited similar sensitivity to phagocytosis or complement-mediated lysis than B5055, unlike the non-encapsulated mutant B5055nm. In vitro, B5055DeltalpxM showed increased permeability of the outer membrane and an increased susceptibility to certain antibacterial peptides suggesting that in vivo attenuation may be due in part to sensitivity to antibacterial peptides present in the lungs of BALB/c mice. These data support the view that lipopolysaccharide acylation plays a important role in providing Gram-negative bacteria some resistance to structural and innate defenses and especially the antibacterial properties of detergents (e.g. bile) and cationic defensins.

Topics: Acylation; Animals; Anti-Bacterial Agents; Bacterial Capsules; Blood Bactericidal Activity; Defensins; Disease Models, Animal; Drug Resistance, Microbial; Humans; Klebsiella Infections; Klebsiella pneumoniae; Lipid A; Mice; Mice, Inbred BALB C; Phagocytosis; Pneumonia, Bacterial; Sepsis

To investigate the vaccine potential of both the Toxoplasma GRA2 and GRA6 antigens, the full length recombinant proteins were produced in Escherichia coli, formulated in MPL adjuvant, and used alone and in combination ("mix"), to immunize CBA/J mice. Although high ratios of specific IgG2a/IgG1 were measured against both proteins, only spleen cells from GRA2-immunized mice and mix-immunized mice produced high amounts of both IFN-gamma and IL-2 upon induction with Toxoplasma gondii Excretory-Secretory Antigens. Intra peritoneal challenge with Toxoplasma cysts resulted in significant reduction of brain cysts in GRA2- and in mix-vaccinated mice only. This study shows the protective efficacy of recombinant GRA2 against chronic infection by T. gondii and confirms the utility of MPL adjuvant in enabling a vaccine candidate to induce a protective Th1 immune response.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Protozoan; Antigens, Protozoan; Brain; Cytokines; Disease Models, Animal; Escherichia coli; Humans; Lipid A; Mice; Mice, Inbred CBA; Protozoan Proteins; Protozoan Vaccines; Th1 Cells; Toxoplasma; Toxoplasmosis, Animal; Vaccines, Subunit; Vaccines, Synthetic

Melioidosis is an emerging disease of humans in Southeast Asia and tropical Australia. The bacterium causing this disease, Burkholderia pseudomallei, is also considered a bioterrorism agent, and as yet there is no licensed vaccine for preventing B. pseudomallei infection. In this study, we evaluated selected proteins (LolC, PotF, and OppA) of the ATP-binding cassette systems of B. pseudomallei as candidate vaccine antigens. Nonmembrane regions of the B. pseudomallei proteins were expressed and purified from Escherichia coli and then evaluated as vaccine candidates in an established mouse model of B. pseudomallei infection. When delivered with the monophosphoryl lipid A-trehalose dicorynomycolate adjuvant, the proteins stimulated antigen-specific humoral and cellular immune responses. Immunization with LolC or PotF protein domains afforded significant protection against a subsequent challenge with B. pseudomallei. The most promising vaccine candidate, LolC, provided a greater level of protection when it was administered with immune-stimulating complexes complexed with CpG oligodeoxynucleotide 10103. Immunization with LolC also protected against a subsequent challenge with a heterologous strain of B. pseudomallei, demonstrating the potential utility of this protein as a vaccine antigen for melioidosis.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Antigens, Bacterial; ATP-Binding Cassette Transporters; Bacterial Vaccines; Burkholderia pseudomallei; Disease Models, Animal; Escherichia coli; Female; Lipid A; Melioidosis; Mice; Mice, Inbred BALB C; Oligodeoxyribonucleotides; Survival Analysis; T-Lymphocytes; Vaccines, Subunit; Vaccines, Synthetic

E5564, a competitive lipid A antagonist, inhibits endotoxin-stimulated inflammation and is under study in patients with sepsis.. We tested whether clinically relevant variables, including the timing of treatment and the route of infection, influenced the effective dosing of E5564 in Escherichia coli-challenged rats.. All E5564 doses (0.3, 1.0, 2.0, and 3.0 mg/kg intravascular bolus followed by 10% of the bolus dose infused hourly for 24 h) administered 1 h before intravascular E. coli challenge similarly reduced the risk of death. Delaying the start of E5564 to 1 or 3 h after intravascular E. coli challenge significantly reduced the beneficial effect of the doses tested. However, increasing the dose of E5564 reversed some loss of efficacy for delayed treatment (P=.004, for increasing benefit with increasing dose at 1 h). During intrabronchial or intraperitoneal (extravascular) E. coli challenge, the pattern of effective E5564 dosing was the inverse of that for intravascular E. coli challenge (P=.001, for the interaction)--lower doses of E5564 were beneficial and higher doses were not (0.03, 0.3, 1.0, 2.0, and 3.0 mg/kg bolus followed by infusion) (P=.05, for decreasing benefit with increasing dose at 1 h).. These findings suggest that, for maximal clinical benefit, E5564 should be given early and that dosing should be adjusted upward for intravascular infection and downward for extravascular infection.

Topics: Animals; Blood Chemical Analysis; Blood Circulation; Colony Count, Microbial; Disease Models, Animal; Endotoxins; Escherichia coli Infections; Leukocyte Count; Lipid A; Lipopolysaccharides; Lung; Random Allocation; Rats; Rats, Sprague-Dawley; Sepsis; Survival Analysis; Time Factors; Tumor Necrosis Factor-alpha

An intranasal vaccine targeting the Bacillus anthracis toxin and vegetative bacterium was tested for the ability to protect immunized rabbits against aerosol B. anthracis spore exposure. Rabbits were vaccinated intranasally with PA-based vaccines formulated as dry powders with or without chitosan (ChiSys, Archimedes Development Limited), a compound that exhibits muco-adhesive properties, or as a liquid. Formulations also contained MPL adjuvant and PA. Some vaccines contained PA conjugated to a 10-mer peptide of the poly-d-glutamic acid capsule of B. anthracis. Rabbits were immunized on days 0 and 28 and aerosol challenged with an average 250LD50 Ames spores on day 85. Serum antibody was measured before and after challenge. Significant anti-PA serum IgG levels were obtained, particularly with use of ChiSys based formulations. PA-Conj induced significant anti-capsule responses, although a formulation containing free capsule peptide did not. All immunized rabbits survived the challenge, but differences in morbidity, as evidenced by anorexia, between vaccine groups were observed. Only rabbits immunized with PA+PA-Conj appeared normal throughout the post-challenge observation period (14 days), while all that received PA with the free capsule peptide appeared ill at times as evidenced by a failure to eat normally. One negative control rabbit received a lower inhaled spore dose (183LD50) and survived the challenge, although it was anorexic post-challenge. It also had a high level of anti-LF antibodies in its convalescent serum (5400 U/ml), indicating an extensive infection. In contrast, 75% of the immunized rabbits had no LF-specific antibody in their post-challenge sera, and the rest had low levels (< or = 138 U/ml), indicating that infections resulting in toxin production were avoided or greatly reduced. Thus, intranasal immunization with a chitosan-based powder vaccine combining PA and capsule epitopes provided superior protection against B. anthracis infection compared to a single antigen (PA) vaccine, as evidenced by a reduction in morbidity and prevention of death.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Aerosols; Animals; Anorexia; Anthrax; Anthrax Vaccines; Antibodies, Bacterial; Antigens, Bacterial; Bacillus anthracis; Bacterial Capsules; Bacterial Toxins; Chitosan; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Immunoglobulin G; Inhalation Exposure; Lipid A; Neutralization Tests; Polyglutamic Acid; Rabbits; Recombinant Fusion Proteins

Using a rabbit model of tuberculous meningitis, we evaluated the protective efficacy of vaccination with the recombinant polyprotein Mtb72F, which is formulated in two alternative adjuvants, AS02A and AS01B, and compared this to vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) alone or as a BCG prime/Mtb72F-boost regimen. Vaccination with Mtb72F formulated in AS02A (Mtb72F+AS02A) or Mtb72F formulated in AS01B (Mtb72F+AS01B) was protective against central nervous system (CNS) challenge with Mycobacterium tuberculosis H37Rv to an extent comparable to that of vaccination with BCG. Similar accelerated clearances of bacilli from the cerebrospinal fluid, reduced leukocytosis, and less pathology of the brain and lungs were noted. Weight loss of infected rabbits was less extensive for Mtb72F+AS02A-vaccinated rabbits. In addition, protection against M. tuberculosis H37Rv CNS infection afforded by BCG/Mtb72F in a prime-boost strategy was similar to that by BCG alone. Interestingly, Mtb72F+AS01B induced better protection against leukocytosis and weight loss, suggesting that the polyprotein in this adjuvant may boost immunity without exacerbating inflammation in previously BCG-vaccinated individuals.

Topics: Adjuvants, Immunologic; Animals; Bacterial Proteins; Body Weight; Central Nervous System; Disease Models, Animal; Drug Combinations; Drug Evaluation, Preclinical; Immunity, Active; Immunization, Secondary; Lipid A; Lung; Mycobacterium bovis; Mycobacterium tuberculosis; Rabbits; Recombinant Proteins; Saponins; Tuberculosis Vaccines; Tuberculosis, Meningeal

Activation of innate immunity using adjuvants that activate Toll-like receptor 4 pathways have great potential for improving the protection induced by parasite vaccines. We investigated protective and therapeutic effects of a vaccine against leishmaniasis containing a combination of an adjuvant synthetic lipid A-analogue, ONO-4007 and Leishmania amazonensis antigens. ONO-4007 was co-injected with soluble and membrane-enriched L. amazonensis-amastigote antigens into BALB/c mice that had either already been infected with 1 x 10(6) L. amazonensis promastigotes (immunotherapy study) or before challenge with the same infectious dose (immunoprophylaxis study). Sixty percent of mice vaccinated before infectious challenge controlled their Leishmania infections - defined by the absence of footpad-swelling and negative Leishmania cultures - compared to 0% of controls, and 40% of mice vaccinated after infection resolved their infections compared to 0% of controls. Protective immunity in both immunoprophylaxis and immunotherapy models was associated with increased protein production of IL-12 and IFN-gamma. These data suggest that vaccination with a combination of ONO-4007 and amastigote antigens of L. amazonensis may be useful for the prevention and treatment of leishmaniasis, and that the protective immunity induced is associated with the production of type-1 cytokines.

Topics: Animals; Antigens, Protozoan; Disease Models, Animal; Female; Interferon-gamma; Interleukin-12; Leishmania; Leishmaniasis, Cutaneous; Lipid A; Mice; Mice, Inbred BALB C; Protozoan Vaccines

Topics: Animals; Disease Models, Animal; Ischemic Preconditioning; Lipid A; Male; MAP Kinase Signaling System; Rats; Rats, Wistar; Reperfusion Injury

Current evidence indicates that the chronic inflammation observed in the intestines of patients with inflammatory bowel disease is due to an aberrant immune response to enteric flora. We have developed a lipid A-mimetic, CRX-526, which has antagonistic activity for TLR4 and can block the interaction of LPS with the immune system. CRX-526 can prevent the expression of proinflammatory genes stimulated by LPS in vitro. This antagonist activity of CRX-526 is directly related to its structure, particularly secondary fatty acyl chain length. In vivo, CRX-526 treatment blocks the ability of LPS to induce TNF-alpha release. Importantly, treatment with CRX-526 inhibits the development of moderate-to-severe disease in two mouse models of colonic inflammation: the dextran sodium sulfate model and multidrug resistance gene 1a-deficient mice. By blocking the interaction between enteric bacteria and the innate immune system, CRX-526 may be an effective therapeutic molecule for inflammatory bowel disease.

Topics: Adjuvants, Immunologic; Animals; Anti-Inflammatory Agents, Non-Steroidal; ATP Binding Cassette Transporter, Subfamily B, Member 1; Caproates; Cells, Cultured; Colitis; Dextran Sulfate; Disease Models, Animal; Female; Glucosamine; HeLa Cells; Humans; Inflammatory Bowel Diseases; Lipid A; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Knockout; Monocytes; Receptors, Immunologic; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

A vaccine trial was conducted to determine the efficacy of a multicomponent candidate vaccine, FALVAC-1, against Plasmodium falciparum in Aotus nancymai monkeys. After two immunizations, animals were challenged intravenously with parasites of the Vietnam Oak Knoll (FVO) strain of P. falciparum. The primary outcome was to determine the protective response of the monkeys to immunization with the FALVAC-1 antigen produced in baculovirus when combined with different adjuvants (alum, QS-21, ASO2a, CRL1005/oil, and CRL1005/saline) as compared with FALVAC-1 with FCA/FIA and antigen alone. When compared with the monkeys immunized with FALVAC-1 alone, FALVAC-1 with FCA/FIA reduced the mean parasite count (to Day 11), reduced the mean accumulated parasitemia (through Day 11), and extended the number of days to treatment. None of the other 5 antigen-adjuvant combinations were able to provide discernable levels of protection based on log(parasitemia) and log(cumulative parasitemia) to Day 11.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Aotidae; Disease Models, Animal; Drug Combinations; Female; Freund's Adjuvant; Hematocrit; Immunization Schedule; Lipid A; Malaria Vaccines; Malaria, Falciparum; Male; Plasmodium falciparum; Polymers; Saponins; Treatment Outcome; Vaccines, Synthetic

Alpha-D-glucopyranose,3-O-decyl-2-deoxy-6-O-[2-deoxy-3-O-[(3R)-3-methoxydecyl]-6-O-methyl-2-[[(11Z)-1-oxo-11-octadecenyl]amino]-4-O-phosphono-beta-D-glucopyranosyl]-2-[(1,3-dioxotetradecyl)amino]-1-(dihydrogen phosphate), tetrasodium salt (E5564) is a second-generation synthetic lipodisaccharide designed to antagonize the toxic effects of endotoxin, a major immunostimulatory component of the outer cell membrane of Gram negative bacteria. In vitro, E5564 dose dependently (nanomolar concentrations) inhibited lipopolysaccharide (LPS)-mediated activation of primary cultures of human myeloid cells and mouse tissue culture macrophage cell lines as well as human or animal whole blood as measured by production of tumor necrosis factor-alpha and other cytokines. E5564 also blocked the ability of Gram negative bacteria to stimulate human cytokine production in whole blood. In vivo, E5564 blocked induction of LPS-induced cytokines and LPS or bacterial-induced lethality in primed mice. E5564 was devoid of agonistic activity when tested both in vitro and in vivo and has no antagonistic activity against Gram positive-mediated cellular activation at concentrations up to 1 microM. E5564 blocked LPS-mediated activation of nuclear factor-kappaB in toll-like receptor 4/MD-2-transfected cells. In a mouse macrophage cell line, activity of E5564 was independent of serum, suggesting that E5564 exerts its activity through the cell surface receptor(s) for LPS, without the need for serum LPS transfer proteins. Similar to (6-O-[2-deoxy-6-O-methyl-4-O-phosphono-3-O-[(R)-3-Z-dodec-5-endoyloxydecl]-2-[3-oxo-tetradecanoylamino]-beta-O-phosphono-alpha-D-glucopyranose tetrasodium salt (E5531), another lipid A-like antagonist, E5564 associates with plasma lipoproteins, causing low concentrations of E5564 to be quantitatively inactivated in a dose- and time-dependent manner. However, compared with E5531, E5564 is a more potent inhibitor of cytokine generation, and higher doses retain activity for durations likely sufficient to permit clinical application. These results indicate that E5564 is a potent antagonist of LPS and lacks agonistic activity in human and animal model systems, making it a potentially effective therapeutic agent for treatment of disease states caused by endotoxin.

Topics: Animals; Blood; Cells, Cultured; Cytokines; Disease Models, Animal; Drosophila Proteins; Drug Interactions; Endotoxins; Escherichia coli; Guinea Pigs; Humans; Lipid A; Lipopolysaccharides; Macrophages; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Monocytes; Rats; Rats, Sprague-Dawley; Receptors, Cell Surface; Shock, Septic; Time Factors; Toll-Like Receptor 4; Toll-Like Receptors; Tumor Necrosis Factor-alpha

C57BL/6 mice were vaccinated with a bacterially expressed and purified polyhistidine-tagged full-length version of the microneme protein NcMIC3 (recNcMIC3) emulsified in Ribi Adjuvant System (RAS). Subsequently, they were challenged by intraperitoneal inoculation of 2 x 10(6) live Neospora caninum tachyzoites. As controls, groups of mice received phosphate-buffered saline (PBS)-RAS alone (adjuvant control) or were treated with PBS before infection (infection control). The protective effect of vaccination was assessed by Neospora-specific polymerase chain reaction (PCR), immunohistochemical investigation of brain tissue, and serological means (enzyme-linked immunosorbent assay). Assessment by PCR performed on DNA from different organs revealed that in all treatment groups parasite DNA could only be detected in brain tissue. According to the PCR results. the recNcMIC3 vaccine conferred protection to 75% of mice (n = 16 in 2 independent experiments), whereas application of PBS-RAS and of PBS alone resulted in protection of 12.5% and 0% of mice, respectively (n = 16 as above). Mice in the PBS-treated infection control group were affected by evident clinical signs of neosporosis starting on day 6 postinfection (p.i.). Conversely, none of the animals treated with either PBS-RAS or recNcMIC3 exhibited any symptoms until day 21 p.i. Immunohistochemical staining of paraffin-embedded brain tissue sections confirmed the protective effect of recNcMIC3 vaccination. Quantitative Neospora-specific real-time PCR revealed that infection intensities were lower in the brain tissues of recNcMIC3-vaccinated mice compared with PBS-RAS-treated adjuvant control mice. Serological analysis showed that the protective effect observed in recNcMIC3-vaccinated mice was associated with a Th2-type IgG1 antibody response directed against native NcMIC3 and a mixed IgG1-IgG2a antibody response directed against the recombinant antigen itself. Taken together, these results demonstrated that recombinant NcMIC3 vaccine confers a significant protectivity against experimentally induced cerebral neosporosis in mice.

Topics: Adhesins, Bacterial; Animals; Antibodies, Protozoan; Antigens, Protozoan; Brain; Brain Diseases; Carrier Proteins; Cell Wall Skeleton; Coccidiosis; Cord Factors; Disease Models, Animal; DNA, Protozoan; Female; Immunoglobulin G; Lipid A; Mice; Mice, Inbred C57BL; Neospora; Polymerase Chain Reaction; Protozoan Proteins; Protozoan Vaccines; Recombinant Proteins; Vaccination; Vaccines, Synthetic

The goal of this project was the evaluation of a novel immunomodulatory adjuvant for human use, OM-174, which is a soluble adjuvant derived from Escherichia coli lipid A. For this study, we used a synthetic peptide, known for its safety and reproducibility and the murine model of BALB/c mice. The long peptide (PbCS 242-310) used corresponds to the C-terminal region of the circumsporozoite protein (CSP) that is the major protein on the surface of Plasmodium sporozoites. Subcutaneous injections of PbCS 242-310 in combination with soluble adjuvant OM-174 induced long lasting peptide-specific antibody titres comparable to those obtained by immunization with incomplete Freund's adjuvant (IFA). The ex vivo evaluation of the CD8(+) T cell response by IFN-gamma ELISPOT assay revealed that the injection of polypeptide with OM-174 adjuvant induced, compared to IFA, a similar and an eight-fold increased frequency of peptide-specific lymphocytes in the draining lymph-nodes and in the spleen, respectively. The CD8(+) T-cells are specific for the sequence PbCS 245-253, a well-known H-2K(d)-restricted CTL epitope, and are cytotoxic as shown in a chromium release assay. Immunization of BALB/c mice with this polypeptide in combination with adjuvant OM-174 conferred a protection after challenge with live Plasmodium berghei sporozoites.The strong antibody and CTL responses observed to a synthetic peptide in mice, the safety profile of the adjuvant and its extensive physico-chemical characterization suggest that OM-174 has a potential use in vaccine formulations for humans.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Protozoan; Antigens, Protozoan; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Humans; Lipid A; Lipopolysaccharides; Malaria; Malaria Vaccines; Mice; Mice, Inbred BALB C; Peptide Fragments; Plasmodium berghei; Protozoan Proteins

Development of an effective vaccine against Leishmania infection is a priority of tropical disease research. We have recently demonstrated protection against Leishmania major in the murine and nonhuman primate models with individual or combinations of purified leishmanial recombinant antigens delivered as plasmid DNA constructs or formulated with recombinant interleukin-12 (IL-12) as adjuvant. In the present study, we immunized BALB/c mice with a recombinant polyprotein comprising a tandem fusion of the leishmanial antigens thiol-specific antioxidant, L. major stress-inducible protein 1 (LmSTI1), and Leishmania elongation initiation factor (LeIF) delivered with adjuvants suitable for human use. Aspects of the safety, immunogenicity, and vaccine efficacy of formulations with each individual component, as well as the polyprotein referred to as Leish-111f, were assessed by using the L. major challenge model with BALB/c mice. No adverse reactions were observed when three subcutaneous injections of the Leish-111f polyprotein formulated with either MPL-squalene (SE) or Ribi 529-SE were given to BALB/c mice. A predominant Th1 immune response characterized by in vitro lymphocyte proliferation, gamma interferon production, and immunoglobulin G2A antibodies was observed with little, if any, IL-4. Moreover, Leish-111f formulated with MPL-SE conferred immunity to leishmaniasis for at least 3 months. These data demonstrate success at designing and developing a prophylactic leishmaniasis vaccine that proved effective in a preclinical model using multiple leishmanial antigens produced as a single protein delivered with a powerful Th1 adjuvant suitable for human use.

Topics: Adjuvants, Immunologic; Animals; Antigens, Protozoan; Antioxidants; Cell Wall Skeleton; Cord Factors; Disease Models, Animal; Female; Heat-Shock Proteins; Leishmania major; Leishmania mexicana; Leishmaniasis, Cutaneous; Lipid A; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Chaperones; Peptide Initiation Factors; Peroxidases; Peroxiredoxins; Polyproteins; Protozoan Proteins; Protozoan Vaccines; T-Lymphocytes; Vaccination; Vaccines, Synthetic

E5564, a synthetic lipid A analogue, is a selective, highly active antagonist of endotoxin-mediated activation of immune cells. Preclinical research has indicated that E5564 can block endotoxin-mediated induction of cytokines and endotoxin or Gram-negative bacterial-induced death in animal models. Recent phase I clinical trials have focused on the ability of E5564 to block responsiveness to endotoxin. This was done in two ways: in vivo challenge of human volunteers with 4 ng/kg endotoxin, and by use of an ex vivo assay which utilizes blood drawn from volunteers administered E5564 and challenged with endotoxin at concentrations that ranged from 50 pg/ml to 10 ng/ml. In vivo, > or = 100 microg of E5564 completely blocked signs, symptoms and cytokines induced by concomitantly-administered endotoxin. In contrast, subjects receiving a 50 microg dose of E5564 demonstrated a graded response; cytokines were inhibited > or = 95%, but many signs and symptoms of endotoxemia were still evident. E5564 demonstrated a long pharmacokinetic half-life (> 30 h); however, ex vivo analysis indicated that while single doses of 350 microg induced a nearly complete block of the effects of 1 ng/ml endotoxin immediately upon E5564 administration, antagonistic activity declined rapidly (t(1/2) < 1 h). Similar results were obtained in vivo using a delayed endotoxin challenge. These results have driven us to examine antagonistic activity of E5564 in vivo and ex vivo after administration by continuous infusion or twice-daily dosing. Results from these multiple-dose studies indicate that under these conditions of administration, plasma levels of E5564 can be predictive of long-term pharmacodynamic activity.

Topics: Animals; Cell Culture Techniques; Cytokines; Disease Models, Animal; Half-Life; Humans; Lipid A; Lipopolysaccharides; Macrophages; Mice

Shigella flexneri is a Gram-negative pathogen that invades and causes inflammatory destruction of the human colonic epithelium, thus leading to bloody diarrhea and dysentery. A type III secretion system that delivers effector proteins into target eukaryotic cells is largely responsible for cell and tissue invasion. However, the respective role of this invasive phenotype and of lipid A, the endotoxin of the Shigella LPS, in eliciting the inflammatory cascade that leads to rupture and destruction of the epithelial barrier, was unknown. We investigated whether genetic detoxification of lipid A would cause significant alteration in pathogenicity. We showed that S. flexneri has two functional msbB genes, one carried by the chromosome (msbB1) and the other by the virulence plasmid (msbB2), the products of which act in complement to produce full acyl-oxy-acylation of the myristate at the 3' position of the lipid A glucosamine disaccharide. A mutant in which both the msbB1 and msbB2 genes have been inactivated was impaired in its capacity to cause TNF-alpha production by human monocytes and to cause rupture and inflammatory destruction of the epithelial barrier in the rabbit ligated intestinal loop model of shigellosis, indicating that lipid A plays a significant role in aggravating inflammation that eventually destroys the intestinal barrier. In addition, neutralization of TNF-alpha during invasion by the wild-type strain strongly impaired its ability to cause rupture and inflammatory destruction of the epithelial lining, thus indicating that TNF-alpha is a major effector of epithelial destruction by Shigella.

Topics: Acylation; Acyltransferases; Animals; Bacterial Proteins; Caco-2 Cells; Disease Models, Animal; Dysentery, Bacillary; Escherichia coli Proteins; Genes, Bacterial; Guinea Pigs; Humans; Intestinal Mucosa; Ligation; Lipid A; Monocytes; Mutagenesis; Rabbits; Shigella flexneri; Tumor Necrosis Factor-alpha; Virulence

Immunity against Mycobacterium tuberculosis depends largely on activation of cell-mediated responses, and gamma interferon has been shown to play a crucial role in this process in both humans and animal models. Since the lung is normally the organ in which infection is initiated and is the major site of pathology, immune responses in the lung play a significant role in restricting initial infection with M. tuberculosis. The aim of the present study was to stimulate efficient immunity in the lung by targeting the gut mucosa. Detoxified monophosphoryl lipid A (MPL) has been shown to be a relatively nontoxic adjuvant which efficiently promotes the induction of type 1 responses when it is given by the traditional subcutaneous route. We have therefore compared subcutaneous immunization of mice to oral immunization by using a model subunit vaccine carrying two immunodominant proteins from M. tuberculosis, in combination with MPL-based adjuvants. While less effective when used to prime a response, a heterologous priming and boosting vaccination strategy employing oral boosting induced significant systemic type 1 responses which equaled and surpassed those attained by subcutaneous immunization protocols. Moreover, the increased immune responses observed correlated with the induction of substantial protection against subsequent aerosol infection with virulent M. tuberculosis at levels comparable to, or better than, those obtained by multiple subcutaneous vaccinations. These results demonstrate that booster vaccinations via mucosal surfaces, by combining efficient subunit vaccines with the potent adjuvant MPL, may be an effective method of addressing some of the shortcomings of current vaccination strategies.

Topics: Acyltransferases; Adjuvants, Immunologic; Administration, Oral; Aerosols; Animals; Antigens, Bacterial; Bacterial Proteins; Bacterial Toxins; BCG Vaccine; Cholera Toxin; Disease Models, Animal; Enterotoxins; Escherichia coli Proteins; Female; Guinea Pigs; Immunization, Secondary; Injections, Subcutaneous; Lipid A; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Recombinant Fusion Proteins; Tuberculosis, Pulmonary; Vaccination; Vaccines, Synthetic

Toll-like receptor 4 (TLR4) mediates lipopolysaccharide (LPS) signaling in a variety of cell types. MD-2 is associated with the extracellular domain of TLR4 and augments TLR4-dependent LPS responses in vitro. We show here that MD-2(-/-) mice do not respond to LPS, do survive endotoxic shock but are susceptible to Salmonella typhimurium infection. We found that in MD-2(-/-) embryonic fibroblasts, TLR4 was not able to reach the plasma membrane and predominantly resided in the Golgi apparatus, whereas TLR4 was distributed at the leading edge surface of cells in wild-type embryonic fibroblasts. Thus, MD-2 is essential for correct intracellular distribution and LPS-recognition of TLR4.

Topics: Animals; Antigens, Surface; B-Lymphocytes; Cells, Cultured; Dendritic Cells; Disease Models, Animal; Drosophila Proteins; Female; Interleukin-12; Interleukin-6; Intracellular Fluid; Lipid A; Lipopolysaccharide Receptors; Lymphocyte Antigen 96; Macrophages; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptors, Cell Surface; Salmonella Infections; Salmonella typhimurium; Shock, Septic; Spleen; Toll-Like Receptor 4; Toll-Like Receptors; Tumor Necrosis Factor-alpha

Development of brain metastases despite extracerebral response to systemic immunotherapy is a common problem in melanoma patients. We have previously described a murine melanoma vaccine of interferon-gamma (IFNgamma)-treated, irradiated syngeneic B16/G3.12 and allogeneic (Cloudman) melanoma cells, plus the adjuvant DETOX, that is protective against subcutaneous (93%) or intracerebral (69%) syngeneic challenge. This study aimed to optimize this vaccine. Groups of nine or 10 mice were immunized five times in 5 weeks with: (i) complete vaccine +/- IFNgamma (VAC+, VAC-); (ii) syngeneic 2 x 106 G3.12 cells plus DETOX (Syn+D), (iii) 2 x 106 allogeneic Cloudman cells plus DETOX (Allo+D); (iv) VAC+ without DETOX (no DETOX); (v) DETOX alone (DETOX); or (vi) phosphate buffered saline (PBS). Mice were challenged subcutaneously with 104 viable G3.12 (or Cloudman cells) and after 35 days intracerebrally with 104 G3.12 cells. Expression of H-2 antigens (measured using fluorescence-activated cell sorting), splenocyte cytotoxicity (measured using 51Cr release) and median overall survival (OAS) were analysed using the log-rank test. VAC+, VAC- and G3.12 mice were equally protected from subcutaneous (s.c.) and intracerebral (i.c.) melanoma challenge (OAS 65 days for s.c., 30 days for i.c.). Protection was less (P < 0.05) in DETOX mice (48 days for s.c.), PBS mice (47 days for s.c., 21 days for i.c.) or no DETOX mice (51 days for s.c.). Allo+D mice showed s.c. (59 days) but not i.c. protection (20 days). IFNgamma incubation did not increase the effect in either the challenge cells or the vaccine cells (P > 0.05). Specific cytotoxicity was seen with G3.12 targets in VAC+ (27%) but not PBS (2%; P < 0.05) mice with equal NK (YAC-1) lysis (10% versus 7%; P< 0.05). Optimal protection against s.c./i.c. experimental murine melanoma was yielded by irradiated syngeneic cells plus DETOX. DETOX alone was not active. Upregulation of H-2 antigens with IFNgamma under these conditions does not augment protection.

Topics: Animals; Autopsy; Brain Neoplasms; Cancer Vaccines; Cytoskeletal Proteins; Cytotoxicity, Immunologic; Disease Models, Animal; Down-Regulation; Drug Combinations; Female; H-2 Antigens; Interferon-gamma; Lipid A; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Skin Neoplasms; Spleen; Survival Rate; Tumor Cells, Cultured; Up-Regulation

Bacterial endotoxin reduces the severity of ventricular arrhythmias which occur when a coronary artery is occluded several hours later. We have now examined in anaesthetised dogs the effects on ischaemia and reperfusion-induced arrhythmias, of a non-toxic derivative component of the endotoxin molecule of the lipid-A (monophosphoryl lipid-A). This was given intravenously, in doses of 10 and 100 microg kg(-1), 24 h prior to coronary artery occlusion. Arrhythmia severity was markedly reduced by monophosphoryl lipid-A. During ischaemia, ventricular premature beats were reduced from 315+/-84 in the vehicle controls to 89+/-60 (with the lower dose of monophosphoryl lipid-A) and 53+/-23 (P<0.05) with the higher dose. The incidence of ventricular tachycardia was reduced from 75% to 25% (P<0.05) and 31% (P<0.05), and the number of episodes of ventricular tachycardia from 13.4+/-4.9 per dog to 1.1+/-1.1 (P<0.05) and 1. 2+/-0.9 (P<0.05) after doses of 10 and 100 microg kg(-1), respectively. The incidence of ventricular fibrillation during occlusion and reperfusion in the control group was 96% (15/16), i.e., only 6% (1/16) dogs survived the combined ischaemia-reperfusion insult. Monophosphoryl lipid-A (100 microg kg(-1)) significantly reduced the incidence of occlusion-induced ventricular fibrillation (from 50% to 7%; P<0.05), and increased survival following reperfusion to 54% (P<0.05). Monophosphoryl lipid-A also significantly reduced ischaemia severity as assessed from ST-segment elevation recorded from epicardial electrodes as well as the degree of inhomogeneity of electrical activation within the ischaemia area. There were no haemodynamic differences prior to coronary occlusion between vehicle controls and monophosphoryl lipid-A-treated dogs. These results demonstrate that monophosphoryl lipid-A reduces arrhythmia severity 24 h after administration. Although the precise mechanisms are still unclear, there is some evidence that nitric oxide and prostanoids (most likely prostacyclin) may be involved because the dual inhibition of nitric oxide synthase and cyclooxygenase enzymes by administration of aminoguanidine and meclofenamate abolished the marked antiarrhythmic protection resulted from monophosphoryl lipid-A treatment 24 h previously.

Topics: Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Arterial Occlusive Diseases; Coronary Vessels; Disease Models, Animal; Dogs; Enzyme Inhibitors; Female; Guanidines; Heart Ventricles; Hemodynamics; Lipid A; Male; Meclofenamic Acid; Myocardial Ischemia; Myocardial Reperfusion Injury; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Prostaglandin-Endoperoxide Synthases; Severity of Illness Index; Tachycardia, Ventricular; Time Factors; Ventricular Fibrillation

The results of this study provide the first evidence that two completely separate vaccine approaches, one based on a subunit vaccine consisting of a mild adjuvant admixed with purified culture filtrate proteins and enhanced by the cytokine interleukin-2 and the second based on immunization with DNA encoding the Ag85A protein secreted by Mycobacterium tuberculosis, could both prevent the onset of caseating disease, which is the hallmark of the guinea pig aerogenic infection model. In both cases, however, the survival of vaccinated guinea pigs was shorter than that conferred by Mycobacterium bovis BCG, with observed mortality of these animals probably due to consolidation of lung tissues by lymphocytic granulomas. An additional characteristic of these approaches was that neither induced skin test reactivity to commercial tuberculin. These data thus provide optimism that development of nonliving vaccines which can generate long-lived immunity approaching that conferred by the BCG vaccine is a feasible goal.

Topics: Acyltransferases; Animals; Antigens, Bacterial; Bacterial Proteins; BCG Vaccine; Disease Models, Animal; Evaluation Studies as Topic; Female; Guinea Pigs; Hypersensitivity, Delayed; Interleukin-2; Lipid A; Lung; Mice; Tuberculosis, Pulmonary; Vaccination; Vaccines, DNA

Polymicrobial sepsis induced by cecal ligation and puncture (CLP) reproduces many of the pathophysiologic features of septic shock. In this study, we demonstrate that mRNA for a broad range of pro- and anti-inflammatory cytokine and chemokine genes are temporally regulated after CLP in the lung and liver. We also assessed whether prophylactic administration of monophosphoryl lipid A (MPL), a nontoxic derivative of lipopolysaccharide (LPS) that induces endotoxin tolerance and attenuates the sepsis syndrome in mice after CLP, would alter tissue-specific gene expression post-CLP. Levels of pulmonary interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), granulocyte colony-stimulating factor (G-CSF), IL-1 receptor antagonist (IL-1ra), and IL-10 mRNA, as well as hepatic IL-1beta, IL-6, gamma interferon (IFN-gamma), G-CSF, inducible nitric oxide synthase, and IL-10 mRNA, were reduced in MPL-pretreated mice after CLP compared to control mice. Chemokine mRNA expression was also profoundly mitigated in MPL-pretreated mice after CLP. Specifically, levels of pulmonary and hepatic macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, MIP-2, and monocyte chemoattractant protein-1 (MCP-1) mRNA, as well as hepatic IFN-gamma-inducible protein 10 and KC mRNA, were attenuated in MPL-pretreated mice after CLP. Attenuated levels of IL-6, TNF-alpha, MCP-1, MIP-1alpha, and MIP-2 in serum also were observed in MPL-pretreated mice after CLP. Diminished pulmonary chemokine mRNA production was associated with reduced neutrophil margination and pulmonary myeloperoxidase activity. These data suggest that prophylactic administration of MPL mitigates the sepsis syndrome by reducing chemokine production and the recruitment of inflammatory cells into tissues, thereby attenuating the production of proinflammatory cytokines.

Topics: Adjuvants, Immunologic; Animals; Cecum; Chemokines; Cytokines; Disease Models, Animal; Gene Expression Regulation; Immune Tolerance; Kinetics; Lipid A; Liver; Lung; Mice; Mice, Inbred C57BL; Neutrophils; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; RNA, Messenger; Sepsis

A novel experimental model of submassive liver necrosis with impaired regeneration has been established. A novel lipid A analogue, FS-112, was injected intravenously into male BALB/c mice, followed 2 days later by a 70% partial hepatectomy. Over the next 9 days, mice became severely jaundiced, with a peak total bilirubin (TBil) concentration of (mean +/- s.d.) 12.9 +/- 2.1 mg/dL 7 days postoperatively. In contrast, the TBil concentration in vehicle-treated mice remained less than 2 mg/dL. Significant elevations of L-alanine:2-oxoglutarate aminotransferase (ALT) were also observed 3-7 days after the operation in mice pretreated with FS-112, compared with mice pretreated with the vehicle. Submassive liver necrosis was observed with extensive mononuclear cell infiltration in mice treated with FS-112 and subjected to partial hepatectomy. Furthermore, both the BrdU and the proliferating cell nuclear antigen (PCNA) labelling index (LI) 1 day following partial hepatectomy in mice pretreated with FS-112 (8.6 +/- 4.3 and 7.9 +/- 4.2%, respectively) were significantly lower than levels in vehicle-treated mice (25.8 +/- 3.8 and 26.5 +/- 10.5%, respectively). The time course of changes in the BrdU LI in liver specimens from mice treated with both FS-112 and partial hepatectomy did not increase, even 3, 5, and 7 days postoperatively. Excellent liver regeneration with a PCNA LI 10-fold higher than the resting level was observed in mice treated with D-galactosamine hydrochloride. These results strongly suggest that this animal model of submassive liver necrosis may be suitable for clarifying the mechanisms of impaired liver cell regeneration often seen in fulminant hepatitis.

Topics: Animals; Bilirubin; Cell Division; Disease Models, Animal; Hepatectomy; Hepatic Encephalopathy; Lipid A; Liver; Liver Regeneration; Male; Mice; Mice, Inbred BALB C; Necrosis; Time Factors

Old and young Balb/c mice, 24--26 and 2--4 months old, respectively, were infected with a 0.1 LD50 of influenza A/Taiwan/1/86 (A/H1N1) virus by small particle aerosol. Lung virus titers were determined 4, 6, 8, 12, and 17 days later. Old mice had significantly higher virus titers than young mice (P < 0.05-0.0001) and shed virus up to Day 17, while young mice were free of virus by Day 12. Splenic MHC class I CD8+ CTL activity (P < 0.08--0.001) and IFN-gamma production (0.1-0.008) measured on Days 8, 12, and 17 were significantly lower among old mice than among young mice. Coadministration of liposomal influenza vaccine with monophosphoryl lipid A (MPL) resulted in enhanced CD8+ CTL response and IFN-gamma production among old mice (35 and 12,000 times, respectively). These results demonstrate that MPL stimulates CTL and Th1 cytokines (IFN-gamma) in aged mice and may serve to reverse age-related CD8+ CTL deficiency and reduce severe influenza disease in elderly human populations.

Topics: Adjuvants, Immunologic; Aging; Animals; Disease Models, Animal; Histocompatibility Antigens Class I; Humans; Influenza A virus; Influenza Vaccines; Influenza, Human; Interferon-gamma; Lipid A; Liposomes; Lung; Mice; Mice, Inbred BALB C; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

The antitumor effect and biological activities of a newly synthesized lipid A analogue (ONO-4007) were investigated in a hamster pancreatic carcinoma model. Marked and dose-dependent inhibition of tumor growth was achieved by i.p. injection twice a week for 3 weeks of 10, 30 or 50 mg/kg of ONO-4007. Endogenous tumor necrosis factor (TNF) activities induced by ONO-4007 were significantly greater in tumor than in serum, spleen and liver. TNF production by macrophages stimulated with ONO-4007 after culture was much greater when culture was performed in the presence of hamster pancreatic carcinoma cells (no cell-to-cell contact). It was further found that the cytotoxic activity of TNF secreted by macrophages cultured with cancer cells was inhibited in the presence of anti-TNF neutralizing antibodies. These findings suggest that ONO-4007 displays antitumor effects by stimulating production of endogenous TNF in tumor macrophages, possibly through activation by soluble macrophage-stimulating factors in cancer cells.

Topics: Animals; Antineoplastic Agents; Cricetinae; Disease Models, Animal; Female; Injections, Intraperitoneal; Lipid A; Macrophages, Peritoneal; Mesocricetus; Neoplasm Transplantation; Pancreatic Neoplasms; Tumor Necrosis Factor-alpha

We studied the role of adhesion molecules in acute lung injury caused by lipopolysaccharide (LPS). Forty-eight female guinea pigs were divided into three groups: saline (n = 12); B464, an LPS antagonist, (0.2 mg/kg i.v.) (n = 12); LPS (0.02 mg/kg i.v.) (n = 12); and LPS + B464 (n = 12). The numbers of polymorphonuclear cells (PMN) in blood sampled over 4 hours were counted. Accumulation of PMNs in the lungs was determined by counting the number of PMNs in lung-tissue samples fixed for light-microscopic examination. The lung wet-to-dry weight ratio and the 125I-albumin tissue-to-plasma ratio were used to assess lung injury. Human umbilical-vein endothelial cells were treated with B464 and then stimulated with either LPS or tumor necrosis factor. Expression of ICAM-1 and ELAM-1 was estimated by enzyme-linked immunosorbent assay. After LPS injection, light microscopy revealed decreases in peripheral PMN counts, and accumulation of PMNs in the lungs. Increases in the two indices of lung injury were also observed. These changes were attenuated by prior treatment with B464. The LPS-induced increases in ICAM-1 and ELAM-1 expression were dose-dependently suppressed by B464. These results suggest that pulmonary accumulation of activated PMNs plays an important role in LPS-induced lung injury.

Topics: Acute Disease; Animals; Cells, Cultured; Disease Models, Animal; E-Selectin; Endothelium, Vascular; Female; Guinea Pigs; Humans; Intercellular Adhesion Molecule-1; Lipid A; Lipopolysaccharides; Lung Diseases

Lipid As from non-toxic bacteria such as Rhodobacter capsulatus and Rhodobacter sphaeroides have been shown to antagonize the immunostimulatory effects of lipid A and LPS from pathogenic bacteria. We have biologically characterized a series of synthetic LPS antagonists including the proposed structures of the lipid A and R. sphaeroides containing fatty acid side chains ester-linked to the disaccharide backbone, as well as an analog of R. capsulatus lipid A containing ether-linked alkyloxy side chains (E5531). In vitro assays utilizing LPS-stimulated human monocytes or whole blood demonstrated that low nanomolar concentrations of E5531 inhibited cellular activation as indicated by decreased release of the cytokines TNF-a, and interleukins-1, 6, and 8. E5531 also inhibited LPS-induced release of cytokines and nitric oxide from murine macrophages. Synthetic antagonists at up to 100 microM were devoid of agonistic activity in murine and human in vitro systems. In vivo, E5531 blocked induction of TNF-a by LPS and reduced LPS-induced lethality in mice. These in vitro and in vivo results indicate that E5531 may have clinical therapeutic utility as an antagonist of endotoxin-mediated morbidity and mortality.

Topics: Animals; Carbohydrate Sequence; Disease Models, Animal; Endotoxins; Humans; In Vitro Techniques; Lipid A; Macrophages; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Monocytes; Nitric Oxide; Shock, Septic; Tumor Necrosis Factor-alpha

The ability of monophosphoryl lipid A (MLA) to provide prophylactic protection against septic shock was evaluated in a mouse model of induced endotoxin hypersensitivity. Treatments of hypersensitized animals with low doses of MLA attenuated endotoxin lethality and endotoxin-mediated liver damage. These effects were related to the ability of MLA to suppress accumulation of TNF-alpha and IFN-gamma in the bloodstream of animals. MLA treatments had only a modest effect in suppressing the accumulation of nitrate in the bloodstream. This implied that MLA did not suppress induction of macrophage and hepatocyte nitric oxide synthetases that contribute to antimicrobial defense and protect against endotoxin-mediated liver damage. The MLA treatments did not appear to compromise inflammatory defenses against local infection since locally recruited leukocytes remained responsive to endotoxin after hypersensitivity had been attenuated. In agreement with these findings, other studies have shown that the induction of endotoxin tolerance by MLA parallels the induction of resistance of animals to lethal challenges with either Gram negative or Gram positive bacteria. As predicted from preclinical studies, human trials of the clinical form of MLA (MPL-immunostimulant) have confirmed that MLA could attenuate systemic responses to endotoxin in normal volunteers, including the attenuation of blood cytokine accumulation and attenuation of symptomatic responses.

Topics: Adjuvants, Immunologic; Animals; Disease Models, Animal; Female; Humans; Interferon-gamma; Lipid A; Lipopolysaccharides; Liver; Macrophages, Peritoneal; Mice; Mice, Inbred ICR; Nitric Acid; Sepsis; Shock, Septic; Tumor Necrosis Factor-alpha

Treatment of log phase cultures of Escherichia coli with cell wall active antibiotics results in increased exposure of immunologically reactive lipid A epitopes of lipopolysaccharide (LPS) and release of soluble LPS into culture supernatants. Comparison of the efficacy of two cell wall active antibiotics, ceftazidime, a penicillin-binding protein 3 selective antibiotic, and imipenem, a penicillin-binding protein 2 selective antibiotic, for their relative efficacy in mediating LPS release indicated quantitative but not qualitative differences, with the former antibiotic manifesting a significantly broader range of concentrations at which LPS release could be demonstrated. Comparison of the relative efficacy of these two antibiotics in a mouse bacteraemia model in which animals were made hypersensitive to the lethal effects of endotoxin by treatment with D-galactosamine indicated that the latter antibiotic may provide a greater level of protection. These studies suggest that the release of endotoxin mediated by antibiotic treatment may contribute to the pathogenesis of disease in infectious due to gram-negative organisms.

Topics: Animals; Ceftazidime; Disease Models, Animal; Escherichia coli; Escherichia coli Infections; Female; Galactosamine; Imipenem; Lipid A; Lipopolysaccharides; Mice; Microbial Sensitivity Tests

The major objective of the present study was to determine the effects of a new endotoxin analogue, monophosphoryl lipid A (MLA), on myocardial infarct size in dogs. A second aim was to determine if potential cardioprotective effects of MLA might be mediated via an enhancement of antioxidant defence mechanisms.. Barbiturate anaesthetised dogs were subjected to 60 min left circumflex coronary artery occlusion followed by 5 h reperfusion. Either of two different doses of MLA (30 and 100 micrograms.kg-1) or an equivalent volume of vehicle were given intravenously 24 h prior to the infarct experiments. Transmural myocardial blood flow was measured at 30 min of occlusion by the radioactive microsphere technique and infarct size was determined at the end of 5 h of reperfusion by triphenyltetrazolium staining. Tissue catalase and myeloperoxidase activities were measured at 5 h of reperfusion as indices of antioxidant activity and neutrophil infiltration, respectively.. There were no significant differences between groups in systemic haemodynamic variables, myocardial oxygen demand, ischaemic bed size, or coronary and collateral blood flow to the ischaemic region. However, administration of MLA produced a marked dose dependent reduction in myocardial infarct size: 19.8(SEM 3.7)% and 14.1(2.5)%, respectively, v 32.7(2.9)% in the vehicle control group, p < 0.05. Pretreatment with either 30 or 100 micrograms.kg-1 of MLA resulted in small increases in tissue catalase activity in the non-ischaemic region of the heart: 0.169(0.033) and 0.197(0.013) K.g-1, respectively, v 0.136(0.013) K.g-1 tissue in the control; however, the increases were not statistically significant by ANOVA. Myeloperoxidase activity in the border zone immediately adjacent to the infarct was markedly decreased in both MLA treated groups: MLA 30 micrograms.kg-1, 2.69(0.82); MLA 100 micrograms.kg-1, 2.49(0.47), v control group, 5.81(1.20) units.g-1 tissue; p < 0.05.. These data are the first to show a marked cardioprotective effect of a lipid A derivative of endotoxin in an in vivo model of myocardial infarction. Although the mechanism responsible for the reduction in infarct size by MLA is unknown, a reduction in neutrophil migration at the site of ongoing tissue injury, the border zone, may be partially responsible.

Topics: Animals; Catalase; Disease Models, Animal; Dogs; Endotoxins; Female; Heart; Lipid A; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Peroxidase

To examine the hemodynamic effects of diphosphoryl lipid A from Rhodopseudomonas sphaeroides and to examine the ability of this substance to induce tolerance to endotoxic shock.. Randomized, prospective, controlled study comparing the hemodynamic actions of R. sphaeroides diphosphoryl lipid A to those effects of lipopolysaccharide form Salmonella minnesota, followed by a prospective, randomized, controlled study comparing pretreatment with R. sphaeroides diphosphoryl lipid A and phosphate-buffered saline in the induction of tolerance to endotoxic shock.. Laboratory of the Section of Critical Care Medicine at a University Hospital.. Male Sprague-Dawley rats.. Eight rats were randomized to receive intravenous R. sphaeroides diphosphoryl lipid A, 0.5 mg/100 g body weight or S. minnesota lipopolysaccharide, 0.5 mg/100 g body weight. Ten rats were then randomized to receive R. sphaeroides diphosphoryl lipid A, 0.5 mg/100 g body weight, or phosphate-buffered saline intravenously 48 hrs before receiving S. minnesota lipopolysaccharide, 5 mg/100 g body weight, by intravenous infusion.. Cardiac index was significantly decreased from baseline in rats treated with lipopolysaccharide; there was no significant change in the R. sphaeroides diphosphoryl lipid A group. Peak circulating tumor necrosis factor (TNF) concentrations in the lipopolysaccharide-treated rats were higher than in R. sphaeroides diphosphoryl lipid A-treated rats (3.1 +/- 1.0 vs. 1.5 +/- 0.4 ng/mL). R. sphaeroides diphosphoryl lipid A significantly attenuated lipopolysaccharide-induced changes in mean arterial pressure and cardiac index. At baseline, there was no significant difference in serum TNF concentrations between rats pretreated with R. sphaeroides diphosphoryl lipid A and those rats pretreated with phosphate-buffered saline. TNF levels peaked at 1 hr post-lipopolysaccharide infusion at 4.3 +/- 0.6 ng/mL in the phosphate-buffered saline group and at 2.0 +/- 0.5 ng/mL in the R. sphaeroides diphosphoryl lipid A group (p < .02). Four of five rats pretreated with R. sphaeroides diphosphoryl lipid A survived endotoxic shock, whereas none of the phosphate-buffered saline-pretreated rats survived (p = .05).. These observations are consistent with previous reports of the limited toxic effects of R. sphaeroides diphosphoryl lipid A and suggest that this molecule retains the ability to induce tolerance to endotoxic shock.

Topics: Animals; Disease Models, Animal; Drug Evaluation, Preclinical; Gram-Negative Bacterial Infections; Hemodynamics; Infusions, Intravenous; Lipid A; Lipopolysaccharides; Male; Random Allocation; Rats; Rats, Sprague-Dawley; Rhodobacter sphaeroides; Salmonella; Shock, Septic; Survival Rate; Time Factors; Tumor Necrosis Factor-alpha

Defibrotide (DEF), a compound previously found to stimulate vascular prostacyclin (PGI2) formation, has been investigated in an experimental model of septic shock. Anesthetized pigs were subjected to i.v. infusion of lipid A (1.5 mg/kg per hr for 4 hr). DEF (50 mg/kg per hr) or vehicle were infused i.v. throughout the experiments, starting 1 hr prior to lipid A. Two out of 7 pigs receiving vehicle survived lipid A infusion for 4 hr, whereas 6 out of 7 DEF treated animals survived this period (P less than 0.05). DEF delayed the shock-induced depression of platelet count and preserved platelet secretory function (collagen-induced ATP-secretion). DEF increased plasma PGI2 by 45% (P less than 0.05) during lipid A infusion and tended to reduce thromboxane levels. DEF did not change eicosanoid formation in sham-shock pigs (n = 4 per group). In vivo treatment with DEF significantly increased the stimulatory effect of bradykinin (1 microM) and arachidonic acid (100 microM) on PGI2 formation ex vivo of mesenteric and iliac artery segments. The improvement of survival in lipid A-induced shock by DEF may be related to an enhancement of vascular PGI2 generation, potentially due to a reduction of shock-induced platelet activation and microcirculatory dysfunction.

Topics: Animals; Arachidonic Acid; Blood Platelets; Bradykinin; Disease Models, Animal; Drug Synergism; Epoprostenol; Lipid A; Platelet Count; Polydeoxyribonucleotides; Shock; Survival Rate; Swine; Thromboxane A2

Immunogenicity of the lipid A component of Haemophilus somnus lipooligosaccharide in cattle and mice was examined after purification, detoxification, and covalent conjugation to a protein carrier. After 2 inoculations, a substantial antibody response was induced in most cattle to lipid A and the protein carrier. To determine whether antibodies to lipid A would be protective, 5 x 10(7) colony-forming units of H somnus strain 649 were administered IV to endotoxin-responsive (C3H/HeN) mice. In one study, 8 of 13 C3H/HeN mice aborted when inoculated. In contrast, abortion did not result when mice were inoculated with the same dose of an isolate of H somnus normally found in the prepuce or with the rough mutant Escherichia coli J5. In addition, endotoxin-nonresponsive (C3H/HeJ) mice were significantly (P = 0.03) more resistant to abortion by strain 649 than were C3H/HeN mice, but inoculated C3H/HeN mice were only slightly more resistant to H somnus abortion, compared with control mice. Although a large antibody response to lipid A was detected, there was no significant difference in the immunized group between mice that aborted and mice that delivered normally. Thus, lipooligosaccharide and other properties of virulent H somnus strains may contribute to abortion in mice.

Topics: Abortion, Veterinary; Animals; Antibodies, Bacterial; Bacterial Vaccines; Cattle; Cattle Diseases; Cricetinae; Disease Models, Animal; Female; Haemophilus; Haemophilus Infections; Hemocyanins; Lipid A; Mesocricetus; Mice; Mice, Inbred C3H; Ovalbumin; Pregnancy; Rats; Rats, Inbred Strains; Vaccines, Synthetic

Various reactions of disseminated intravascular coagulation (DIC) were experimentally induced by infusion of thrombokinase in rats, by administration of endotoxin in rabbits and pigs and by infusion of adrenaline and thrombin in dogs. Low plasma concentrations of recombinant hirudin (r-hirudin; 20-200 ng/ml) were sufficient for the inhibition of the different triggering mechanisms. The studies on the pharmacological profile of r-hirudin in DIC therapy confirm the efficacy of this specific tight-binding thrombin inhibitor.

Topics: Animals; Disease Models, Animal; Disseminated Intravascular Coagulation; Dogs; Endotoxins; Epinephrine; Factor Xa; Factor Xa Inhibitors; Female; Fibrinolytic Agents; Hemodynamics; Hirudin Therapy; Hirudins; Lipid A; Male; Platelet Count; Rabbits; Rats; Rats, Inbred Strains; Recombinant Proteins; Species Specificity; Swine; Thrombin; Thrombolytic Therapy; Thrombosis; Viscera

To study the role of antibodies in promoting survival during gram-negative bacterial sepsis, we have developed several murine monoclonal antibodies (MAbs). One MAb (5B10) reacted in an enzyme-linked immunosorbent assay with only a single organism (Escherichia coli 0111:B4), while the other (8A1) reacted to all gram-negative whole-cell and lipopolysaccharide (LPS) antigens examined. Either 5B10 MAb, 8A1 MAb, or sterile saline solution was administered intravenously to outbred male Swiss-Webster mice immediately before one of three challenges: (1) viable bacteria intravenously, (2) viable bacteria with hemoglobin intraperitoneally, or (3) intravenous actinomycin D plus LPS. 5B10 MAb provided significant protection against either an E. coli 0111:B4 bacterial or LPS challenge but not against any other organism or type of LPS. 8A1 MAb provided protection against several challenge bacteria (intravenously or intraperitoneally) and against all types of LPS studied except Pseudomonas aeruginosa LPS. A higher dose (2 mg) of cross-reactive antibody (8A1 MAb) was required to produce protection when compared with the type-specific protection produced with 5B10 MAb (0.1 mg). Although ideal antibody therapy would consist of directing a specific MAb against a single microorganism, the acute nature of the disease process and time required to prepare reagents may preclude the use of type-specific MAbs. We believe that the cross-reactive and cross-protective capacity of 8A1 MAb or a similar MAb may be useful in averting the lethal effects of clinical gram-negative bacterial sepsis and warrants testing in the clinical setting.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Cross Reactions; Disease Models, Animal; Endotoxins; Enzyme-Linked Immunosorbent Assay; Escherichia coli Infections; Lipid A; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Peritonitis; Sepsis