Influenza "Trains" the Host for Enhanced Susceptibility to Secondary Bacterial Infection.

We previously reported that the Toll-like receptor 4 (TLR4) antagonist Eritoran blocks acute lung injury (ALI) therapeutically in mouse and cotton rat models of influenza. However, secondary (2°) bacterial infection following influenza virus infection is associated with excess morbidity and mortality. Wild-type (WT) mice infected with mouse-adapted influenza A/Puerto Rico/8/34 virus (PR8) and, 7 days later, with Streptococcus pneumoniae serotype 3 (Sp3) exhibited significantly enhanced lung pathology and lethality that was reversed by Eritoran therapy after PR8 infection but before Sp3 infection. Cotton rats infected with nonadapted pH1N1 influenza virus and then superinfected with methicillin-resistant Staphylococcus aureus also exhibited increased lung pathology and serum high-mobility-group box 1 (HMGB1) levels, both of which were blunted by Eritoran therapy. In mice, PR8 infection suppressed Sp3-induced CXCL1 and CXCL2 mRNA, reducing neutrophil infiltration and increasing the bacterial burden, all of which were reversed by Eritoran treatment. While beta interferon (IFN-ß)-deficient (IFN-ß-/-) mice are highly susceptible to PR8, they exhibited delayed death upon Sp3 superinfection, indicating that while IFN-ß was protective against influenza, it negatively impacted the host response to Sp3 IFN-ß-treated WT macrophages selectively suppressed Sp3-induced CXCL1/CXCL2 transcriptionally, as evidenced by reduced recruitment of RNA polymerase II to the CXCL1 promoter. Thus, influenza establishes a "trained" state of immunosuppression toward 2° bacterial infection, in part through the potent induction of IFN-ß and its downstream transcriptional regulation of chemokines, an effect reversed by Eritoran.IMPORTANCE Enhanced susceptibility to 2° bacterial infections following infection with influenza virus is a global health concern that accounts for many hospitalizations and deaths, particularly during pandemics. The complexity of the impaired host immune response during 2° bacterial infection has been widely studied. Both type I IFN and neutrophil dysfunction through decreased chemokine production have been implicated as mechanisms underlying enhanced susceptibility to 2° bacterial infections. Our findings support the conclusion that selective suppression of CXCL1/CXCL2 represents an IFN-ß-mediated "training" of the macrophage transcriptional response to TLR2 agonists and that blocking of TLR4 therapeutically with Eritoran after influenza virus infection reverses this suppression by blunting influenza-induced IFN-ß.

Serum High-Mobility-Group Box 1 as a Biomarker and a Therapeutic Target during Respiratory Virus Infections.

Host-derived "danger-associated molecular patterns" (DAMPs) contribute to innate immune responses and serve as markers of disease progression and severity for inflammatory and infectious diseases. There is accumulating evidence that generation of DAMPs such as oxidized phospholipids and high-mobility-group box 1 (HMGB1) during influenza virus infection leads to acute lung injury (ALI). Treatment of influenza virus-infected mice and cotton rats with the Toll-like receptor 4 (TLR4) antagonist Eritoran blocked DAMP accumulation and ameliorated influenza virus-induced ALI. However, changes in systemic HMGB1 kinetics during the course of influenza virus infection in animal models and humans have yet to establish an association of HMGB1 release with influenza virus infection. To this end, we used the cotton rat model that is permissive to nonadapted strains of influenza A and B viruses, respiratory syncytial virus (RSV), and human rhinoviruses (HRVs). Serum HMGB1 levels were measured by an enzyme-linked immunosorbent assay (ELISA) prior to infection until day 14 or 18 post-infection. Infection with either influenza A or B virus resulted in a robust increase in serum HMGB1 levels that decreased by days 14 to 18. Inoculation with the live attenuated vaccine FluMist resulted in HMGB1 levels that were significantly lower than those with infection with live influenza viruses. RSV and HRVs showed profiles of serum HMGB1 induction that were consistent with their replication and degree of lung pathology in cotton rats. We further showed that therapeutic treatment with Eritoran of cotton rats infected with influenza B virus significantly blunted serum HMGB1 levels and improved lung pathology, without inhibiting virus replication. These findings support the use of drugs that block HMGB1 to combat influenza virus-induced ALI.IMPORTANCE Influenza virus is a common infectious agent causing serious seasonal epidemics, and there is urgent need to develop an alternative treatment modality for influenza virus infection. Recently, host-derived DAMPs, such as oxidized phospholipids and HMGB1, were shown to be generated during influenza virus infection and cause ALI. To establish a clear link between influenza virus infection and HMGB1 as a biomarker, we have systematically analyzed temporal patterns of serum HMGB1 release in cotton rats infected with nonadapted strains of influenza A and B viruses and compared these patterns with a live attenuated influenza vaccine and infection by other respiratory viruses. Towards development of a new therapeutic modality, we show herein that blocking serum HMGB1 levels by Eritoran improves lung pathology in influenza B virus-infected cotton rats. Our study is the first report of systemic HMGB1 as a potential biomarker of severity in respiratory virus infections and confirms that drugs that block virus-induced HMGB1 ameliorate ALI.

HPI/AMF inhibition halts the development of the aggressive phenotype of breast cancer stem cells.

Cancer stem cells are responsible for tumor recurrence and metastasis. A new highly reproducible procedure for human breast cancer MCF-7 stem cells (BCSC) isolation and selection was developed by using a combination of hypoxia/hypoglycemia plus taxol and adriamycin for 24h. The BCSC enriched fraction (i) expressed (2-15 times) the typical stemness protein markers CD44+, ALDH1A3 and Oct 3/4; (ii) increased its clonogenicity index (20-times), invasiveness profile (>70%), migration capacity (100%) and ability to form mammospheres, compared to its non-metastatic MCF-7 counterpart. This isolation and selection protocol was successful to obtain stem cell enriched fractions from A549, SiHa and medulloblastoma cells. Since the secretion of HPI/AMF cytokine seems involved in metastasis, the effects of erytrose-4-phosphate (E4P) and 6-phosphogluconate (6PG), potent HPI inhibitors, on the acquisition of the breast stem cell-like phenotype were also evaluated. The presence of E4P during the BCSC selection deterred the development of the stemness phenotype, whereas both extracellular E4P (5-250nM) and 6PG (1µM) as well as siRNA HPI/AMF depressed the BCSC invasiveness ability (>90%), clonogenicity index (>90%) and contents (50-96%) of stemness (CD44, ALDH1A), pluripotency (p38 MAPK, Oct3/4, wnt/ß-catenin) and EMT (SNAIL, MMP-1, vimentin) markers. The cytokine inhibitor repertaxin (10nM) or the anti-IL-8 or anti-TGF-ß monoclonal antibodies (10µg/mL) did not significantly affect the BCSC metastatic phenotype. E4P also diminished (75%) the formation and growth of MCF-7 stem cell mammospheres. These results suggested that E4P by directly interacting with extracellular HPI/AMF may be an effective strategy to deter BCSC growth and progression.

The Toll-Like Receptor 4 Antagonist Eritoran Protects Mice from Lethal Filovirus Challenge.

The 2013-2016 outbreak of Ebola virus (EBOV) in West Africa, which has seen intermittent reemergence since it was officially declared over in February of 2016, has demonstrated the need for the rapid development of therapeutic intervention strategies. Indirect evidence has suggested that the EBOV infection shares several commonalities associated with the onset of bacterial sepsis, including the development of a "cytokine storm." Eritoran, a Toll-like receptor 4 (TLR4) antagonist, was previously shown to result in protection of mice against lethal influenza virus infection. Here, we report that eritoran protects against the lethality caused by EBOV and the closely related Marburg virus (MARV) in mice. Daily administration of eritoran reduced clinical signs of the disease and, unexpectedly, resulted in reduced viral titers. Analysis of peripheral blood indicated that eritoran reduced granulocytosis despite an apparent increase in the percentage of activated neutrophils. Surprisingly, the increased survival rate and reduced viremia were not accompanied by increased CD3+ T lymphocytes, as lymphopenia was more pronounced in eritoran-treated mice. Overall, a global reduction in the levels of multiple cytokines, chemokines, and free radicals was detected in serum, suggesting that eritoran treatment may alleviate the severity of the "cytokine storm." Last, we provide compelling preliminary evidence suggesting that eritoran treatment may alter the kinetics of cytokine responses. Hence, these studies are the first to demonstrate the role of TLR4 in the pathogenesis of EBOV disease and indicate that eritoran is a prime candidate for further evaluation as a clinically viable therapeutic intervention strategy for EBOV and MARV infections.IMPORTANCE A hallmark of bacterial sepsis is the uncontrolled activation of the TLR4 pathway, which is the primary cause of the pathological features associated with this disease. Considering the importance of TLR4 signaling in bacterial sepsis and the remarkable pathological similarities associated with infections caused by filoviruses Ebola virus (EBOV) and Marburg virus (MARV), we assessed the ability of eritoran, a TLR4 antagonist, to protect mice against these viruses. Here, we show that eritoran effectively promotes survival of mice of filovirus infection, as 70% and 90% of mice receiving daily eritoran treatment survived lethal EBOV and MARV infections, respectively. Eritoran treatment resulted in a remarkable global reduction of inflammatory mediators, which is suggestive of the mechanism of action of this therapeutic treatment. These studies are the first to show the critical importance of the TLR4 pathway in the pathogenesis of filovirus infection and may provide a new avenue for therapeutic interventions.

[Effect of pioglitazone on the expression of TLR4 in renal tissue of diabetic rats].

OBJECTIVE: To investigate the effect of pioglitazone on the expression of Toll-like receptor 4 (TLR4) in renal tissue of diabetic rats. METHODS: The male Sprague Dawlry rats (n=57) were randomly divided into the control group (n=9) and experimental group (n=48). The experimental rats were injected introperatoneally with 50 mg/kg streptozotocin (STZ) and the controls were given sodium citrate buffer instead. Then the experimental rats were randomized into five groups: pioglitazone 5 mg/(kg.d), pioglitazone 10 mg/(kg.d), pioglitazone 5 mg/(kg.d) plus TLR4-specific antagonist Eritoran 5 mg/(kg.d), pioglitazone 10 mg/(kg.d) plus TLR4-specific antagonist Eritoran 5 mg/(kg.d), non-intervented group, with 5 rats in each group. The pioglitazone was administrated intragastrically, and normal saline was given to the contro group in the same way. Five weeks after pioglitazone administration, Eritoran was injected intraperitoneally for consecutive one week. At the end of the eighth week, 24-hour microalbuminuria and the serum concentrations of C-reactive protein (CRP) were determined by radioimmunoassay. Expressions of TLR4 and peroxisome proliferator-activated receptor γ (PPARγ) in kidney were determined by Western blotting and immunohistochemistry. RESULTS: Compared with the control group, the 24-hour urine protein, plasma concentration of CRP, the expression of TLR4 in kidney increased significantly in the experimental groups (P<0.05). Furthermore, these indicators in all intervention groups obviously decreased compared with the non-intervention group (P<0.05). In addition, the expression of TLR4 in high-dose pioglitazone significantly decreased compared with low-dose pioglitazone (P<0.05). The study also found that high-dose pioglitazone and TLR4 antagonist Eritoran could reduce the expression of TLR4 in the diabetic rats, but the difference from the group of low-dose pioglitazone plus Eritoran was not significant statistically (P>0.05). CONCLUSION: Pioglitazone may exert the anti-inflammatory action by decreasing the expression of TLR4 in renal tissue and regulating the balance between proinflammatory and anti-inflammatory.

Safety, pharmacokinetics and pharmacodynamics of four-hour intravenous infusions of eritoran in healthy Japanese and Caucasian men.

Eritoran, a synthetic analogue of lipid A, has been shown to bind to TLR4/MD-2 complex and thereby block the interaction of endotoxins with TLR4. We report here the results of a study conducted to assess the single-dose safety and tolerability, as well as the pharmacokinetics and pharmacodynamics, of eritoran infusion in Japanese and Caucasian healthy adult men. Sixty-four men (aged 20-45 years; body mass index 18-30 kg/m(2)) were randomized into four groups: 4-mg total dose (six Japanese and six Caucasian men); 12-mg total dose (12 Japanese and 12 Caucasian men); 28-mg total dose (six Japanese and six Caucasian men); and placebo (eight Japanese and eight Caucasian men). Eritoran in single doses up to 28 mg over 4 h was well tolerated, with no apparent ethnic differences noted. Plasma concentrations were slightly higher in Japanese versus Caucasian men; these differences were not significant after adjustment for differences in body mass (clearance: approximately 1.2 ml/h/kg; volume of distribution at steady state: approximately 0.07 l/kg). The ex vivo endotoxin inhibitory activity of eritoran was similar in Japanese and Caucasian men. The data do not indicate any need for clinical dose adjustment for possible ethnic-based differences in drug distribution or metabolism.

Toll-like receptor-4 antagonist eritoran tetrasodium for severe sepsis.

The human innate immune system initiates inflammation in response to bacterial molecules, particularly Gram-negative bacterial endotoxin. The steps by which endotoxin exposure leads to systemic inflammation include binding to Toll-like receptor-4 that specifically recognizes endotoxin and subsequently triggers cellular and molecular inflammatory responses. Severe sepsis is a systemic inflammatory response to infection that induces organ dysfunction and threatens a person's survival. Severe sepsis is frequently associated with increased blood levels of endotoxin. It is a significant medical problem that effects approximately 700,000 patients every year in the USA, resulting in 250,000 deaths. Eritoran tetrasodium is a nonpathogenic analog of bacterial endotoxin that antagonizes inflammatory signaling by the immune receptor Toll-like receptor-4. Eritoran is being evaluated for the treatment of patients with severe sepsis.

Influence of plasma cholesterol and triglyceride concentrations and eritoran (E5564) micelle size on its plasma pharmacokinetics and ex vivo activity following single intravenous bolus dose into healthy female rabbits.

PURPOSE: Eritoran (E5564) is a glycophospholipid that acts as a toll-like receptor 4 (TLR4) antagonist that is being tested as a treatment for severe sepsis and septic shock. In the blood, eritoran binds to plasma lipoproteins altering its pharmacokinetic and pharmacodynamic (PD) effects in vivo. The purpose of this study was to determine the influence of changes in plasma cholesterol and triglyceride concentrations on the plasma pharmacokinetics and ex vivo activity of eritoran following single intravenous bolus dosing of eritoran to healthy female rabbits fed either a regular chow diet or a cholesterol-enriched diet. This was done with eritoran administered as stable micelle formulations of mean hydrodynamic diameters of 8 or 27 nm). METHODS: Female New Zealand White rabbits were fed a standard diet for 7 days and then randomly assigned either a regular chow diet [regular-diet (n = 9)] or a cholesterol-enriched diet [cholesterol-diet (n = 12)] for an additional 7 days. Following feeding of these diets a single intravenous bolus dose of eritoran (0.5 mg/kg) formulated into either "small micelles" (8 nm in diameter) or "large micelles" (27 nm in diameter) was administered to regular-fed and cholesterol-fed rabbits. Serial blood samples were obtained prior to eritoran administration and at the following times post injection: 0.083 (5 min), 1, 2, 4, 8, 10, 24, 48 and 72 h. Plasma was analyzed for eritoran concentrations using LC/MS/MS. Total plasma cholesterol (TC) and triglyceride (TG) levels were quantified using enzymatic kits. Plasma eritoran pharmacokinetic (PK) parameters were estimated by non-compartmental analysis using the WinNonlin nonlinear estimation program. To analyze PD activity, whole blood obtained at 0.083 (5 min), 2, 24, 48 and 72 h following eritoran administration was assessed for ex vivo activity by measuring the ability of 1 and 10 ng/ml LPS to elicit TNF-alpha release. RESULTS: Total plasma cholesterol and triglyceride levels were significantly higher in cholesterol-fed rabbits compared to the rabbits fed a regular chow diet. Diet had no effect on the estimated plasma PK parameters. However, PD activity of both small and large micelle eritoran as measured by an ex vivo challenge dose of 1 ng/ml LPS was reduced in blood of cholesterol-fed rabbits compared to normal-fed rabbits. Comparison of PK parameters for small and large micelles indicated that small micelles had increased AUC(0-72 h), decreased plasma clearance and increased initial concentration (measured at 5 min post administration) compared to the large micelle formulation. Consistent with this observation, eritoran formulated into small micelles had significantly greater ex vivo activity than large micelles and was independent of TC and TG concentrations. CONCLUSIONS: These findings suggest that plasma pharmacokinetics and activity of eritoran maybe influenced by eritoran micelle size and plasma TC and TG concentrations.

Cytosolic energy reserves determine the effect of glycolytic sugar phosphates on sarcoplasmic reticulum Ca2+ release in cat ventricular myocytes.

Localization of glycolytic enzymes in close proximity to Ca(2+) transport systems of the sarcoplasmic reticulum (SR) in cardiac cells suggests an important functional role for glycolysis in intracellular [Ca(2+)] regulation and, consequently, excitation-contraction coupling. Here, we investigated the mechanisms of regulation of SR Ca(2+) release by glycolytic sugar phosphate intermediates in cat ventricular myocytes. Experiments with permeabilized myocytes revealed that with normal cytosolic energy reserves (mm: ATP 5, ADP 0.01, phosphocreatine (CrP) 10) fructose-1,6-bisphosphate (FBP; 1 mm) and fructose-6-phosphate (F6P; 1 mm) caused a transient increase of Ca(2+) spark frequency by 62 and 42%, respectively. This effect of sugar phosphates was associated with a 13% decrease in SR Ca(2+) load. Pretreatment of the cells with an inhibitor of glycolysis, iodoacetate (IAA; 0.5 mm), did not prevent the effects of FBP and F6P on Ca(2+) sparks. Recording of single ryanodine receptor (RyR) channel activity indicated that FBP and F6P significantly increased RyR open probability. Reduction of cytosolic energy reserves decreased Ca(2+) spark activity. Increasing [ADP] to 0.4 mm or removal of CrP ([ATP] was kept constant) caused a slowly developing decrease of Ca(2+) spark frequency by 29 and 42%, respectively. Changing [ADP] and [CrP] simultaneously decreased Ca(2+) spark frequency by 66%. This inhibition of Ca(2+) sparks was associated with a 40% decrease in SR Ca(2+) load. The subsequent addition of FBP (1 mm) partially restored Ca(2+) spark frequency and SR Ca(2+) load. This recovery of Ca(2+) sparks was blocked completely by IAA. These data suggest that at physiological ATP, ADP and CrP levels accumulation of sugar phosphates from glycolysis can stimulate SR Ca(2+) release. This effect does not require the activity of downstream glycolytic enzymes, but rather is the result of direct activation of RyRs. However, under conditions associated with depletion of cellular energy reserves (e.g. myocardial ischaemia), ATP generated from glycolysis may play an important role in maintaining myocardial Ca(2+) homeostasis by improving SR Ca(2+) uptake.

Dentine hypersensitivity.

Reports received from 32 dentists on the effect of a complex mixture of calcium sucrose phosphate and calcium orthophosphate used as a gel, toothpaste, or slurry in relieving pain in hypersensitive dentine show, in 137 patients, complete relief in 112. It was found that in 54 patients the prior use of stannous fluoride prophylactic paste was beneficial.

Effect of the interferon inducer, dextran phosphate, on influenza virus infection in mice.

Intraperitoneal administration of dextran phosphate (MW 40,000) or DP 40, an interferon inducer, was shown to increase the survival rate of mice infected with 10 LD50 dose of influenza A2 virus (H2N2). In the treated mice, a 1-day delay was evident in the virus growth in lung, and production of HAI antibody, when compared to the nontreated or dextran-treated controls. More significant was the 2-day delay in the development of lung consolidation, which leads to 40% survival of the treated mice. Mediation of the protection by the interferon induced by DP 40 was indicated.