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When facing a viral infection, the organism has to put in place a number of defense mechanisms in order to clear the pathogen from the cell. At the early phase of this preparation for fighting against the invader, the innate immune response is triggered by the sensing of danger signals. Among those molecular cues, double-stranded RNA (dsRNA) is a very potent inducer of different reactions at the cellular level that can ultimately lead to cell death. Using a genome-wide screening approach, we set to identify genes involved in dsRNA entry, sensing, and apoptosis induction in human cells. This allowed us to determine that the heparan sulfate pathway and the conserved oligomeric Golgi complex are key determinants allowing entry of both dsRNA and viral nucleic acid leading to cell death. Double-stranded RNA (dsRNA) is the hallmark of many viral infections. dsRNA is produced either by RNA viruses during replication or by DNA viruses upon convergent transcription. Synthetic dsRNA is also able to mimic viral-induced activation of innate immune response and cell death. In this study, we employed a genome-wide CRISPR-Cas9 loss-of-function screen based on cell survival in order to identify genes implicated in the host response to dsRNA. By challenging HCT116 human cells with either synthetic dsRNA or Sindbis virus (SINV), we identified the heparan sulfate (HS) pathway as a crucial factor for dsRNA entry, and we validated SINV dependency on HS. Interestingly, we uncovered a novel role for COG4, a component of the conserved oligomeric Golgi (COG) complex, as a factor involved in cell survival to both dsRNA and SINV in human cells. We showed that COG4 knockout led to a decrease of extracellular HS that specifically affected dsRNA transfection efficiency and reduced viral production, which explains the increased cell survival of these mutants. IMPORTANCE When facing a viral infection, the organism has to put in place a number of defense mechanisms in order to clear the pathogen from the cell. At the early phase of this preparation for fighting against the invader, the innate immune response is triggered by the sensing of danger signals. Among those molecular cues, double-stranded RNA (dsRNA) is a very potent inducer of different reactions at the cellular level that can ultimately lead to cell death. Using a genome-wide screening approach, we set to identify genes involved in dsRNA entry, sensing, and apoptosis induction in human cells. This allowed us to determine that the heparan sulfate pathway and the conserved oligomeric Golgi complex are key determinants allowing entry of both dsRNA and viral nucleic acid leading to cell death.

Purpose Emergence of multidrug-resistant strains in intensive care units has renewed interest in colistin, which often remains the only available antimicrobial agent active against resistant Pseudomonas aeruginosa . The aim of this study is to compare lung tissue deposition and antibacterial efficiency between nebulized and intravenous administration of colistin in piglets with pneumonia caused by P. aeruginosa . Methods In ventilated piglets, colistimethate was administered 24 h following bronchial inoculation of Pseudomonas aeruginosa (minimum inhibitory concentration of colistin = 2 μg ml −1 ) either by nebulization (8 mg kg −1 every 12 h, n  = 6) or by intravenous infusion (3.2 mg kg −1 every 8 h, n  = 6). All piglets were killed 49 h after inoculation. Colistin peak lung tissue concentrations and lung bacterial burden were assessed on multiple post mortem subpleural lung specimens. Results Median colistin peak lung concentration following nebulization was 2.8 μg g −1 (25–75% interquartile range = 0.8–13.7 μg g −1 ). Colistin was undetected in lung tissue following intravenous infusion. In the aerosol group, peak lung tissue concentrations were significantly greater in lung segments with mild pneumonia (median = 10.0 μg g −1 , 25–75% interquartile range = 1.8–16.1 μg g −1 ) than in lung segments with severe pneumonia (median = 1.2 μg g −1 , 25–75% interquartile range = 0.5–3.3 μg g −1 ) ( p  < 0.01). After 24 h of treatment, 67% of pulmonary segments had bacterial counts <10 2  cfu g −1 following nebulization and 28% following intravenous administration ( p  < 0.001). In control animals, 12% of lung segments had bacterial counts <10 2  cfu g −1 49 h following bronchial inoculation. Conclusion Nebulized colistin provides rapid and efficient bacterial killing in ventilated piglets with inoculation pneumonia caused by Pseudomonas aeruginosa .

Monge's disease is characterized by an excessive erythrocytosis, frequently associated with pulmonary hypertension, in high-altitude dwellers. It has a considerable impact on public health in high-altitude regions. A preliminary study demonstrated the efficiency of acetazolamide (Acz) (250 mg/d for 3 wk) in reducing serum erythropoietin and hematocrit. Evaluate the efficacy and tolerance of a 6-month treatment with 250 mg Acz that could be chronically implemented and its effects on pulmonary artery pressure and cardiac function. A two-phase study was performed in patients (hematocrit > or = 63%) from Cerro de Pasco, Peru (4,300 m). First phase: a double-blind, placebo-controlled study in 55 patients who received a single dose of either 250 mg Acz (n = 40) or placebo (n = 15) by daily oral administration for 12 weeks. Second phase (open label): after a 4-week washout period, all patients received 250 mg Acz for 12 weeks. Hematocrit, blood gases, clinical outcome, and pulmonary artery circulation were evaluated. First phase: Acz decreased by 44% the number of polycythemic subjects (P = 0.02), decreased hematocrit from 69 to 64% (P < 0.001), and increased arterial O(2) pressure from 42 to 45 mm Hg (P < 0.001). No severe adverse effect or hypokalemia was recorded. The second phase reproduced the effects observed during the first phase, without cumulative effects on hematocrit. A 4-week washout restored basal hematocrit. Only patients who received Acz for 6 months showed a clear reduction in pulmonary vascular resistance. Acz reduces erythrocytosis and improves pulmonary circulation in Monge's disease without adverse effects. Its implementation as a chronic treatment for this disease appears efficient and safe.

Purpose Ventilator-associated pneumonia caused by Pseudomonas aeruginosa with impaired sensitivity to ceftazidime is frequent in critically ill patients. The aim of the study was to compare lung tissue deposition and antibacterial efficiency between nebulized and intravenous administrations of ceftazidime in ventilated piglets with pneumonia caused by Pseudomonas aeruginosa with impaired sensitivity to ceftazidime. Methods Ceftazidime was administered 24 h following the intra-bronchial inoculation of Pseudomonas aeruginosa (minimum inhibitory concentration = 16 μg ml −1 ), either by nebulization (25 mg kg −1 every 3 h, n  = 6) or by continuous intravenous infusion (90 mg kg −1 over 24 h after an initial rapid infusion of 30 mg kg −1 , n  = 6). Four non-treated inoculated animals served as controls. All piglets were killed 48 h (intravenous and control groups) or 51 h (aerosol group) after inoculation. Lung tissue concentrations and lung bacterial burden were assessed on multiple post-mortem sub-pleural lung specimens [(lower limit of quantitation = 10 2 colony forming unit (cfu g −1 )]. Results Ceftazidime trough lung tissue concentrations following nebulization were greater than steady-state lung tissue concentrations following continuous intravenous infusion [median and interquartile range, 24.8 (12.6–59.6) μg g −1 vs. 6.1 (4.6–10.8) μg g −1 ] ( p  < 0.001). After 24 h of ceftazidime administration, 83% of pulmonary segments had bacterial counts <10 2  cfu g −1 following nebulization and only 30% following intravenous administration ( p  < 0.001). In control animals, 10% of lung segments had bacterial counts <10 2  cfu g −1 48 h following bronchial inoculation. Conclusion Nebulized ceftazidime provides more efficient bacterial killing in ventilated piglets with pneumonia caused by Pseudomonas aeruginosa with impaired sensitivity to ceftazidime.

Chronic mountain sickness or Monge's disease is characterized by an excessive polycythemia in high-altitude dwellers, with a prevalence of 5 to 18% above 3,200 m. To date, no pharmacologic treatment is available. We evaluated the efficacy of acetazolamide in the treatment of chronic mountain sickness and the importance of nocturnal hypoxemia in its pathophysiology. A double-blind placebo-controlled study was performed in three groups of patients from Cerro de Pasco, Peru (4,300 m), treated orally for 3 weeks with placebo (n = 10), 250 mg of acetazolamide (n = 10), or 500 mg of acetazolamide (n = 10), daily. Acetazolamide decreased hematocrit by 7.1% (p < 0.001) and 6.7% (p < 0.001), serum erythropoietin by 67% (p < 0.01) and 50% (p < 0.001), and serum soluble transferrin receptors by 11.1% (p < 0.05) and 3.4% (p < 0.001), and increased serum ferritin by 540% (p < 0.001) and 134% (p < 0.001), for groups treated with 250 and 500 mg of acetazolamide, respectively. Acetazolamide (250 mg) increased nocturnal arterial O(2) saturation by 5% (p < 0.01) and decreased mean nocturnal heart rate by 11% (p < 0.05) and the number of apnea-hypopnea episodes during sleep by 74% (p < 0.05). The decrease in erythropoietin was attributed mainly to the acetazolamide-induced increase in ventilation and arterial O(2) saturation. Acetazolamide, the first efficient pharmacologic treatment of chronic mountain sickness without adverse effects, reduces hypoventilation, which may be accentuated during sleep, and blunts erythropoiesis. Its low cost may allow wide development with a considerable positive impact on public health in high-altitude regions.

Background: The aim of this study was to compare the bactericidal activity of cefepime plus amikacin against experimental pneumonia induced by a stably derepressed cephalosporinase-producing Enterobacter cloacae strain in immunocompetent and leucopenic rats. Methods: Sixty Wistar rats were used. Leucopenia was induced in half of them by a single intravenous administration of 30 mg/kg cyclophosphamide, while the remaining rats received the same volume of saline. All rats were infected 96 h later by tracheal instillation of 8 log 10 colony-forming units of E. cloacae. Twelve rats (6 immunocompetent and 6 leucopenic) were sacrificed 6 h later to assess the initial bacterial burden to the lungs. Then, the remaining 48 rats received a combination of 60 mg/kg cefepime twice a day and 25 mg/kg amikacin once a day given intraperitoneally or the same volume of saline. Six rats per group (leucopenic or not, treated or not) were sacrificed 12 and 30 h after therapy started. Results: Spontaneous bacterial clearance with time was observed only in immunocompetent rats. Compared to untreated animals, antibiotic administration induced a decrease in lung bacterial titres in immunocompetent and leucopenic rats. The difference was statistically significant only in leucopenic rats. Conclusions: The use of leucopenic rats reduced spontaneous bacterial clearance in the lungs and increased the bactericidal effect of the antibiotic combination and ultimately the confidence in the reliability of the results.