Cell Plasticity in a Mouse Model of Benign Prostate Hyperplasia Drives Amplification of Androgen-Independent Epithelial Cell Populations Sensitive to Antioxidant Therapy.

Benign prostate hyperplasia (BPH) is caused by the nonmalignant enlargement of the transition zone of the prostate gland, leading to lower urinary tract symptoms. Although current medical treatments are unsatisfactory in many patients, the limited understanding of the mechanisms driving disease progression prevents the development of alternative therapeutic strategies. The probasin-prolactin (Pb-PRL) transgenic mouse recapitulates many histopathological features of human BPH. Herein, these alterations parallel urodynamic disturbance reminiscent of lower urinary tract symptoms. Single-cell RNA-sequencing analysis of Pb-PRL mouse prostates revealed that their epithelium mainly includes low-androgen signaling cell populations analogous to Club/Hillock cells enriched in the aged human prostate. These intermediate cells are predicted to result from the reprogramming of androgen-dependent luminal cells. Pb-PRL mouse prostates exhibited increased vulnerability to oxidative stress due to reduction of antioxidant enzyme expression. One-month treatment of Pb-PRL mice with anethole trithione (ATT), a specific inhibitor of mitochondrial ROS production, reduced prostate weight and voiding frequency. In human BPH-1 epithelial cells, ATT decreased mitochondrial metabolism, cell proliferation, and stemness features. ATT prevented the growth of organoids generated by sorted Pb-PRL basal and LSCmed cells, the two major BPH-associated, androgen-independent epithelial cell compartments. Taken together, these results support cell plasticity as a driver of BPH progression and therapeutic resistance to androgen signaling inhibition, and identify antioxidant therapy as a promising treatment of BPH.

Genome-Wide CRISPR-Cas9 Screen Reveals the Importance of the Heparan Sulfate Pathway and the Conserved Oligomeric Golgi Complex for Synthetic Double-Stranded RNA Uptake and Sindbis Virus Infection.

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.

[To have and have not, RNA interference as an antiviral defense system in mammals]. / En avoir ou pas, l'interférence par l'ARN comme défense antivirale chez les mammifères.

RNA silencing is a small RNA based mechanism regulating gene expression and involved in many biological processes in most eukaryotes. In plants, nematodes and arthropods, this mechanism participates to antiviral defense. In mammals, although the RNA silencing machinery is present and needed for the microRNA pathway, its importance as an antiviral defense is still debated. In recent years, several studies have attempted to answer to the question of whether RNA silencing as an antiviral pathway is retained in mammals. However, these studies did not provide a clear answer yet. In this review, we will present the arguments for and against a relevant antiviral role of RNA interference (RNAi) in mammals, by discussing examples of active and functional mammalian antiviral RNAi in specific cell types and/or in specific conditions.

Contributions and lessons learned from virology in contemporary biology.

Virology is a young discipline at the origin of discoveries that revolutionized our vision of biology. Often associated to pathological studies, this science raises more fundamental questions and brings molecular tools required for cellular manipulation. If viruses are considered as our enemies, they are used, sometimes as a last attempt, against multidrug resistant bacteria or to treat cancer.

How to solve the problem of spontaneous bacterial clearance when testing new antibiotic treatment: results on experimental pneumonia due to a derepressed cephalosporinase-producing Enterobacter cloacae.

Because the magnitude of spontaneous bacterial clearance can be similar or even higher than treatment effect, depending upon experimental model and bacterial strain used, this work investigated the value of rendering rats immunosuppressed to facilitate bacterial implantation and reduce spontaneous bacterial clearance. In a first step, rats received a single intravenous cyclophosphamide dose 4 days before infection. Three different doses were tested: 10, 20, and 40 mg/kg. After modeling with NONMEM V, the cyclophosphamide dose required to maintain white blood cell count <1.0 × 10(3)/µL from day 4 to day 5 was 30 mg/kg. In a second step, influence of immunosuppression on lung bacterial titers was characterized. Rats were given one of the three intravenous cyclophosphamide doses (0, 10, 30 mg/kg), and after 4 days, they were infected by tracheal injection of 8.9 ± 0.1 log10 cfu Enterobacter cloacae before being sacrificed at different times. Bacteria in homogenized lungs were quantitatively cultured on Drigalski agar. Bacterial lung count was closely influenced by the grade of induced leukopenia. A single intravenous 30 mg/kg cyclophosphamide dose 4 days before infection suppressed the spontaneous clearance of E. cloacae for at least 30 h without significantly increasing animal mortality; this result seems to be linked to a white blood cell count maintained lower than 1.0 × 10(3)/µL for all the time. This modified animal model could be contributive in the evaluation of antibacterial agents, especially to simulate the behavior of intensive care unit immunocompromised patients.

Animal model methodology: immunocompetent or leucopenic rats, which is the best? Results from a model of experimental pneumonia due to derepressed cephalosporinase-producing Enterobacter cloacae.

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.

Nebulized and intravenous colistin in experimental pneumonia caused by Pseudomonas aeruginosa.

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 microg 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 microg g(-1) (25-75% interquartile range = 0.8-13.7 microg 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 microg g(-1), 25-75% interquartile range = 1.8-16.1 microg g(-1)) than in lung segments with severe pneumonia (median = 1.2 microg g(-1), 25-75% interquartile range = 0.5-3.3 microg 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.

Nebulized ceftazidime in experimental pneumonia caused by partially resistant Pseudomonas aeruginosa.

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 microg 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) microg g(-1) vs. 6.1 (4.6-10.8) microg 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.

Acetazolamide for Monge's disease: efficiency and tolerance of 6-month treatment.

RATIONALE: 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. OBJECTIVES: 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. METHODS: 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. MEASUREMENTS AND MAIN RESULTS: 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. CONCLUSIONS: 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.

Acetazolamide: a treatment for chronic mountain sickness.

RATIONALE: 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. OBJECTIVES: We evaluated the efficacy of acetazolamide in the treatment of chronic mountain sickness and the importance of nocturnal hypoxemia in its pathophysiology. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Differential diffusions of indinavir and lopinavir in genital secretions of human immunodeficiency virus-infected women.

Plasma and cervicovaginal secretion (CVS) samples were collected from 19 human immunodeficiency virus type 1-infected women on lopinavir- or indinavir-containing regimens. Lopinavir and indinavir were detectable in 29 and 93% of CVS samples, respectively, a finding that may be ascribed to these drugs' differences in protein binding and pK(a). The relationship between lopinavir and indinavir pharmacodynamics and viral evolution in the female genital tract should be assessed over time.

Efficacy of beta-lactams for treating experimentally induced pneumonia due to a carbapenem-hydrolyzing metallo-beta-lactamase-producing strain of Pseudomonas aeruginosa.

A rat pneumonia model was established with a Pseudomonas aeruginosa strain that produced the plasmid-encoded metallocarbapenemase VIM-2. A significant decrease in lung bacterial titers was observed when imipenem, cefepime, ceftazidime, and piperacillin-tazobactam were given at the highest doses recommended for humans, despite their high MICs. Aztreonam at high doses produced a similar decrease in bacterial titers.

Antimicrobial susceptibility test: from bacterial population analysis to therapy.

The statistical methods and parameters commonly used to define bacterial susceptibility to antibiotics in vitro such as MIC(50), linear regression or others, usually lead to a considerable loss of information: they do not take into account the heterogeneity of the bacterial population. In contrast, multivariate data analyses are more adapted to the description of biological systems. In this way, a population of a given bacterial species can be separated into homogenous classes corresponding to the different sensitivity and resistance phenotypes. The applications of this mathematical approach include: (i) a new model for more relevant interpretation of antimicrobial susceptibility test results; (ii) numerical estimation of breakpoints having a known risk; (iii) calibration of a technique relative to a reference technique; (iv) detection of strains with new phenotypes; (v) in vitro evaluation of the activity of new compounds.

Estimation of pharmacokinetic parameters by orthogonal regression: comparison of four algorithms.

The contribution of non-linear orthogonal regression for estimation of individual pharmacokinetic parameters when drug concentrations and sampling times are subject to error was studied. The first objective was to introduce and compare four numerical approaches that involve different degrees of approximation for parameter estimation by orthogonal regression. The second objective was to compare orthogonal with non-orthogonal regression. These evaluations were based on simulated data sets from 300 'subjects', thereby enabling precision and accuracy of parameter estimates to be determined. The pharmacokinetic model was a one-compartment open model with first-order absorption and elimination rates. The inter-individual coefficients of variation (CV) of the pharmacokinetic parameters were in the range 33-100%. Eight measurement-error models for times and concentrations (homo- or heteroscedastic with constant CV) were considered. Accuracy of the four algorithms was very close in almost all instances (typical bias, 1-4%). Precision showed three expected trends: root mean squared error (RMSE) increased when the residual error was larger or the number of observations was smaller, and it was highest for the absorption rate constant and common error variance. Overall, RMSE ranged from 5 to 40%. It was found that the simplest algorithm for othogonal regression performed as well as the more complicated approaches. Errors in sampling time resulted in an increased bias and imprecision in individual parameter estimates (especially for k(a) in our example) and in common error variance when the estimation method did not take into account these errors. In this situation, use of orthogonal regression resulted in smaller bias and better precision.

Pharmacokinetics of teicoplanin during plasma exchange.

OBJECTIVE: To study the elimination of teicoplanin during plasma exchange, a procedure currently used to treat a variety of disorders involving immune complexes. Teicoplanin is a glycopeptide antibiotic that exhibits a long terminal half-life (100-150 h) and is highly bound to plasma proteins (unbound fraction (fu)=0.2). METHODS: Twelve adults with systemic polyarteritis nodosa, cryoglobulinemia-induced vasculitis or dysglobulinemic neuropathy undergoing plasma exchange were studied. Each patient received intravenous teicoplanin, 6 mg/kg body weight, immediately before plasma exchange. Plasma was assayed for teicoplanin by high pressure liquid chromatography. RESULTS: A high level of protein binding of teicoplanin was measured within this patient population (98%). The mean quantity of teicoplanin eliminated (+/-SD) was 74.6+/-34.6 mg. The mean drug fraction eliminated by plasma exchange (+/-SD) was 19.5+/-5.6%. Mean fu value as determined by ultrafiltration (+/-SD) was 2.2+/-1.7%. CONCLUSIONS: These results show that plasma exchange influences teicoplanin pharmacokinetics, with a clinically significant quantity being eliminated. If trough teicoplanin concentrations of around 10 mg/L are desired, it is recommended that teicoplanin dosage be supplemented or given after plasma exchange.