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Respiratory syncytial virus (RSV) disease has no effective treatment. JNJ-53718678 is a fusion inhibitor with selective activity against RSV. After confirmation of RSV infection or 5 days after inoculation with RSV, participants (n = 69) were randomized to JNJ-53718678 75 mg (n = 15), 200 mg (n = 17), 500 mg (n = 18), or placebo (n = 17) orally once daily for 7 days. Antiviral effects were evaluated by assessing RSV RNA viral load (VL) area under the curve (AUC) from baseline (before the first dose) until discharge, time-to-peak VL, duration of viral shedding, clinical symptoms, and quantity of nasal secretions. Mean VL AUC was lower for individuals treated with different doses of JNJ-53718678 versus placebo (203.8-253.8 vs 432.8 log10 PFUe.hour/mL). Also, mean peak VL, time to peak VL, duration of viral shedding, mean overall symptom score, and nasal secretion weight were lower in each JNJ-53718678-treated group versus placebo. No clear exposure-response relationship was observed. Three participants discontinued due to treatment-emergent adverse events of grade 2 and 1 electrocardiogram change (JNJ-53718678 75 mg and 200 mg, respectively) and grade 2 urticaria (placebo). JNJ-53718678 at all 3 doses substantially reduced VL and clinical disease severity, thus establishing clinical proof of concept and the compound's potential as a novel RSV treatment. ClinicalTrials.gov: NCT02387606; EudraCT number: 2014-005041-41.

Background Longitudinal respiratory syncytial virus (RSV) dynamics have not been well studied despite the existence of factors favoring prolonged RSV replication including high mutation rates allowing rapid evolution and potential escape from immune control. We therefore measured viral load in previously RSV-naive infants over prolonged time spans. Methods During 2014–2015, quantitative nasal aspirates were collected from 51 RSV-PCR+ infants. Multiple parallel assessments of viral loads were quantified at each collected time point using a well-validated real-time quantitative reverse transcriptase polymerase chain reaction assay. After observing viral load rebound phenomenon in some infants, the viral dynamics of 27 infants with sufficient longitudinal viral load data points were analyzed using the pre-defined criteria for viral rebound. Additional analyses were performed comparing age with viral rebound, viral clearance rates, and viral load area-under-the-curve (AUC VL ). Results The 51 infants (303 nasal aspirate samples; mean of 5.9 per patient) exhibited slower than expected viral clearance. Lower age trended toward slower viral clearance and greater AUC VL . Six infants had detectable viral loads ≥1 month after symptom onset. Ten of twenty-seven evaluable subjects exhibited viral rebound and this rebound was age-dependent ( P =0.0259). All but one rebounder were <70 days old. Conclusion Infants struggle to control primary RSV infections allowing prolonged viral replication and previously undescribed viral rebound; likely representing viral mutational immune escape.

Evidence suggests that β-lactam monotherapy of streptococcal infections may incite stronger inflammation and is inferior to combination therapy with macrolides. We hypothesized that use of macrolides alone or in combination with a β-lactam for group B streptococcal (GBS) sepsis would improve outcomes by reducing inflammation. TNF-α was measured from supernatants of RAW 264.7 cells stimulated with GBS isolates, in presence of four treatment regimens: ampicillin alone, azithromycin alone, or combination of azithromycin plus ampicillin. Mouse model of GBS sepsis was developed and treated with same four regimens. Clinical sepsis scores were monitored; serum cytokines (TNF-α, IL-6, IL-10) and chemokines (MIP-1α) were measured at the end. GBS isolates exposed to azithromycin or combination (compared to ampicillin alone) stimulated less TNF production in vitro. In the murine sepsis model, mortality was lower along with decreased sepsis scores in mice treated with combination therapy. Mean serum IL-6 was lower in mice treated with azithromycin alone (66±52 pg/ml) or combination of ampicillin plus azithromycin (52±22 pg/ml) compared to ampicillin alone (260±160 pg/ml) (p<0.005). Combination therapy of ampicillin+azithromycin improved outcomes in a murine GBS sepsis model; this therapeutic approach deserves additional study.

Whether p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascades are required for inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) accumulation in RAW 264.7 murine macrophages exposed to lipopolysaccharide (LPS) plus recombinant interferon-γ (rIFN-γ) was investigated. By use of Western blotting for iNOS detection and ELISA for quantitation of TNF secretion, three selective inhibitors of these pathways were tested (the p38 inhibitors SB202190 and SB203580 and the MEK 1,2/ERK inhibitor PD98059). Dose-related inhibition of iNOS production was demonstrated when inhibitors were added 1 h before, simultaneously with, or 1 h after LPS plus rIFN-γ stimulation. In contrast, inhibition of TNF secretion was observed only when cells were preincubated with these agents. Thus, both the p38 and ERK pathways are involved in the up-regulation of iNOS and TNF production by murine macrophages, and specific inhibitors of these pathways block macrophage iNOS production even when added 1h after activation of these cells.

Background Infections caused by community-associated strains of methicillin-resistant Staphylococcus aureus (CA-MRSA) are associated with a marked and prolonged host inflammatory response. In a sepsis simulation model, we tested whether the anesthetic ketamine inhibits the macrophage TNF response to antibiotic-exposed CA-MRSA bacteria via its antagonism of N-methyl-D-aspartate (NMDA) receptors. RAW264.7 cells were stimulated for 18 hrs with 10 5 to 10 7 CFU/mL inocula of either of two prototypical CA-MRSA isolates, USA300 strain LAC and USA400 strain MW2, in the presence of either vancomycin or daptomycin. One hour before bacterial stimulation, ketamine was added with or without MK-801 (dizocilpine, a chemically unrelated non-competitive NMDA receptor antagonist), APV (D-2-amino-5-phosphono-valerate, a competitive NMDA receptor antagonist), NMDA, or combinations of these agents. Supernatants were collected and assayed for TNF concentration by ELISA. Results RAW264.7 cells exposed to either LAC or MW2 in the presence of daptomycin secreted less TNF than in the presence of vancomycin. The addition of ketamine inhibited macrophage TNF secretion after stimulation with either of the CA-MRSA isolates (LAC, MW2) in the presence of either antibiotic. The NMDA inhibitors, MK-801 and APV, also suppressed macrophage TNF secretion after stimulation with either of the antibiotic-exposed CA-MRSA isolates, and the effect was not additive or synergistic with ketamine. The addition of NMDA substrate augmented TNF secretion in response to the CA-MRSA bacteria, and the addition of APV suppressed the effect of NMDA in a dose-dependent fashion. Conclusions Ketamine inhibits TNF secretion by MRSA-stimulated RAW264.7 macrophages and the mechanism likely involves NMDA receptor antagonism. These findings may have therapeutic significance in MRSA sepsis.

The role of p38- and extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathways in the up-regulation of inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) production in macrophages stimulated with Streptococcus pneumoniae was examined. Inhibitors of p38 kinases effected significant decreases in the accumulation of iNOS protein in macrophages challenged with pneumococcal cell wall preparations or antibiotickilled pneumococci, even when added up to 6 h after bacterial challenge. In contrast, ERK pathway inhibitors failed to inhibit pneumococcus-induced iNOS protein accumulation. ERK pathway inhibitors significantly reduced TNF secretion when added at the same time as pneumococcal challenge, and inhibitors of both ERK and p38 pathways reduced TNF secretion when added to the macrophages 1 h before stimulation. These data confirm the importance of the p38 and ERK MAP kinase pathways in macrophage activation by bacterial products but indicate that these 2 kinase pathways regulate different macrophage responses in a temporally distinct manner.

The abilities of pentoxifylline and recombinant interleukin-10 (rIL-10) to inhibit tumor necrosis factor (TNF) and inducible nitric oxide synthase (iNOS) production in RAW264.7 murine macrophages were compared. Pentoxifylline consistently inhibited the accumulation of both TNF and iNOS in a dose-dependent manner whether the stimulus was bacterial lipopolysaccharide (LPS), recombinant interferon-γ (rIFN-γ), or LPS plus rIFN-γ. Similarly, rIL-10 consistently reduced TNF production by cells stimulated with LPS, rIFN-γ, or LPS plus rIFN-γ. However, rIL-10 weakly inhibited LPS-induced iNOS production but failed to block (and often augmented) rIFN-γ-induced iNOS production. Combinations of pentoxifylline and rIL-10 led to additive or synergistic inhibition of TNF but not iNOS production; in fact, rIL-10 appeared to interfere with the ability of pentoxifylline to block iNOS accumulation. These data suggest that combinations of antiinflammatory agents may have unanticipated effects on inflammatory mediator production.

The temporal requirements for tyrosine phosphorylation in the induction of tumor necrosis factor (TNF) and inducible nitric oxide synthase (iNOS) were compared in the murine macrophage cell line RAW 264.7. Preincubation of RAW 264.7 cells with herbimycin A or genistein (but not with either of three tyrphostins tested) significantly blocked TNF and iNOS production on exposure of these cells to combinations of lipopolysaccharide (LPS) and interferon-γ (IFN-γ). The addition of either genistein or herbimycin A to RAW 264.7 cell cultures 1–6 h after stimulation with LPS and IFN-γ had little or no effect on TNF production but markedly inhibited iNOS protein accumulation. Together these data indicate that tyrosine kinase inhibitors block iNOS production at a point well downstream of the initial wave of LPS- and IFN-γ-mediated protein tyrosine phosphorylation.

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that appears to play a significant role in the development of neonatal chronic lung disease (CLD). Inflammation and CLD are also associated with respiratory tract colonization with genital mycoplasmas. The possible protective roles of surfactant in mitigating the inflammatory response to these microbes were investigated. Murine RAW 264.7 macrophages were preincubated with an exogenous surfactant and exposed overnight to sterile media, lipopolysaccharide (LPS), Mycoplasma hominis, or Ureaplasma urealyticum. Macrophages released TNF-α in response to challenge with LPS, U. urealyticum, and M. hominis in a concentration-dependent fashion. Surfactant suppressed LPS and M. hominis induced TNF-α production in a dose-dependent manner but suppressed U. urealyticum—mediated TNF-α production only at the higher dose tested. Similar effects were seen in hyperoxia (95% O2). Thus, exogenous bovine surfactant significantly inhibits the production of TNF-α by murine macrophages stimulated with genital mycoplasmas and bacterial LPS.

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