Association between a rapid diagnostic test to detect methicillin-resistant Staphylococcus Aureus pneumonia and decreased vancomycin use in a medical intensive care unit over a 30-month period.

Vancomycin overuse is common, yet few data are available regarding how to improve stewardship of this antibiotic. We identify an association between use of a PCR assay to rule out MRSA pneumonia and a significant, sustained decrease in average vancomycin days of therapy over a 30-month period.

Rapid Detection of Methicillin-Resistant Staphylococcus aureus in BAL: A Pilot Randomized Controlled Trial.

BACKGROUND: Guidelines recommend empirical vancomycin or linezolid for patients with suspected pneumonia at risk for methicillin-resistant Staphylococcus aureus (MRSA). Unneeded vancomycin or linezolid use may unnecessarily alter host flora and expose patients to toxicity. We therefore sought to determine if rapid testing for MRSA in BAL can safely decrease use of vancomycin or linezolid for suspected MRSA pneumonia. METHODS: Operating characteristics of the assay were initially validated against culture on residual BAL. A prospective, unblinded, randomized clinical trial to assess the effect of antibiotic management made on the basis of rapid diagnostic testing (RDT) compared with usual care was subsequently conducted, with primary outcome of duration of vancomycin or linezolid administration. Secondary end points focused on safety. RESULTS: Sensitivity of RPCR was 95.7%, with a negative likelihood ratio of 0.04 for MRSA. The clinical trial randomized 45 patients: 22 to antibiotic management made on the basis of RDT and 23 to usual care. Duration of vancomycin or linezolid administration was significantly reduced in the intervention group (32 h [interquartile range, 22-48] vs 72 h [interquartile range, 50-113], P < .001). Proportions with complications and length of stay trended lower in the intervention group. Hospital mortality was 13.6% in the intervention group and 39.1% for usual care (95% CI of difference, -3.3 to 50.3, P = .06). Standardized mortality ratio was 0.48 for the intervention group and 1.18 for usual care. CONCLUSIONS: A highly sensitive BAL RDT for MRSA significantly reduced use of vancomycin and linezolid in ventilated patients with suspected pneumonia. Management made on the basis of RDT had no adverse effects, with a trend to lower hospital mortality. TRIAL REGISTRY: ClinicalTrials.gov; No. NCT02660554; URL: www.clinicaltrials.gov.

Multidimensional Assessment of the Host Response in Mechanically Ventilated Patients with Suspected Pneumonia.

Rationale: The identification of informative elements of the host response to infection may improve the diagnosis and management of bacterial pneumonia. Objectives: To determine whether the absence of alveolar neutrophilia can exclude bacterial pneumonia in critically ill patients with suspected infection and to test whether signatures of bacterial pneumonia can be identified in the alveolar macrophage transcriptome. Methods: We determined the test characteristics of alveolar neutrophilia for the diagnosis of bacterial pneumonia in three cohorts of mechanically ventilated patients. In one cohort, we also isolated macrophages from alveolar lavage fluid and used the transcriptome to identify signatures of bacterial pneumonia. Finally, we developed a humanized mouse model of Pseudomonas aeruginosa pneumonia to determine if pathogen-specific signatures can be identified in human alveolar macrophages. Measurements and Main Results: An alveolar neutrophil percentage less than 50% had a negative predictive value of greater than 90% for bacterial pneumonia in both the retrospective (n = 851) and validation cohorts (n = 76 and n = 79). A transcriptional signature of bacterial pneumonia was present in both resident and recruited macrophages. Gene signatures from both cell types identified patients with bacterial pneumonia with test characteristics similar to alveolar neutrophilia. Conclusions: The absence of alveolar neutrophilia has a high negative predictive value for bacterial pneumonia in critically ill patients with suspected infection. Macrophages can be isolated from alveolar lavage fluid obtained during routine care and used for RNA-Seq analysis. This novel approach may facilitate a longitudinal and multidimensional assessment of the host response to bacterial pneumonia.

Strong impact of sulfotransferases on DNA adduct formation by 4-aminobiphenyl in bladder and liver in mice.

Bladder cancer risk is 3-4 times higher in men than women, but the reason is poorly understood. In mice, male bladder is also more susceptible than female bladder to 4-aminobiphenyl (ABP), a major human bladder carcinogen; however, female liver is more susceptible than male liver to ABP. We investigated the role of sulfotransferase (Sult) in gender-related bladder and liver susceptibility to ABP. Sulfation reactions of aromatic amine bladder carcinogens catalyzed by Sult may generate highly unstable and toxic metabolites. Therefore, liver Sult may decrease bladder exposure to carcinogens by promoting their toxic reactions in the liver. Notably, the expression of several liver Sults is suppressed by androgen in male mice. Here, we show that two Sults are critical for gender-related bladder susceptibility to ABP in mice. We measured tissue level of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP), a principal ABP-DNA adduct, as readout of tissue susceptibility to ABP. We identified Sutl1a1 and to a lesser extent Sult1d1 as Sults that promote dG-C8-ABP formation in hepatic cells. In mice, gender gap in bladder susceptibility to ABP was narrowed by knocking out Sult1a1 and was almost totally eliminated by knocking out both Sutl1a1 and Sult1d1. This was accompanied by dramatic decrease in ABP genotoxicity in the liver (>97%). These results show the strong impact of the Sults on bladder and liver susceptibility to a human carcinogen. Because liver expression of both Sult1a1 and Sutl1d1 is suppressed by androgen in male mice, our results suggest that androgen renders bladder more exposed to ABP in male mice by suppressing Sult-mediated ABP metabolism in liver, which increases bladder delivery of carcinogenic metabolites.

Hyperdynamic left ventricular ejection fraction in the intensive care unit.

INTRODUCTION: Limited information exists on the etiology, prevalence, and significance of hyperdynamic left ventricular ejection fraction (HDLVEF) in the intensive care unit (ICU). Our aim in the present study was to compare characteristics and outcomes of patients with HDLVEF with those of patients with normal left ventricular ejection fraction in the ICU using a large, public, deidentified critical care database. METHODS: We conducted a longitudinal, single-center, retrospective cohort study of adult patients who underwent echocardiography during a medical or surgical ICU admission at the Beth Israel Deaconess Medical Center using the Multiparameter Intelligent Monitoring in Intensive Care II database. The final cohort had 2867 patients, of whom 324 had HDLVEF, defined as an ejection fraction >70%. Patients with an ejection fraction <55% were excluded. RESULTS: Compared with critically ill patients with normal left ventricular ejection fraction, the finding of HDLVEF in critically ill patients was associated with female sex, increased age, and the diagnoses of hypertension and cancer. Patients with HDLVEF had increased 28-day mortality compared with those with normal ejection fraction in multivariate logistic regression analysis adjusted for age, sex, Sequential Organ Failure Assessment score, Elixhauser score for comorbidities, vasopressor use, and mechanical ventilation use (odds ratio 1.38, 95% confidence interval 1.039-1.842, p =0.02). CONCLUSIONS: The presence of HDLVEF portended increased 28-day mortality, and may be helpful as a gravity marker for prognosis in patients admitted to the ICU. Further research is warranted to gain a better understanding of how these patients respond to common interventions in the ICU and to determine if pharmacologic modulation of HDLVEF improves outcomes.

The inverse relationship between bladder and liver in 4-aminobiphenyl-induced DNA damage.

Bladder cancer risk is significantly higher in men than in women. 4-Aminobiphenyl (ABP) is a major human bladder carcinogen from tobacco smoke and other sources. In mice, male bladder is more susceptible to ABP-induced carcinogenesis than female bladder, but ABP is more carcinogenic in the livers of female mice than of male mice. Here, we show that castration causes male mice to acquire female phenotype regarding susceptibility of bladder and liver to ABP. However, spaying has little impact on organ susceptibility to ABP. Liver UDP-glucuronosyltransferases (UGTs) are believed to protect liver against but sensitize bladder to ABP, as glucuronidation of ABP and its metabolites generally reduces their toxicity and promotes their elimination via urine, but the metabolites are labile in urine, delivering carcinogenic species to the bladder. Indeed, liver expression of ABP-metabolizing human UGT1A3 transgene in mice increases bladder susceptibility to ABP. However, ABP-specific liver UGT activity is significantly higher in wild-type female mice than in their male counterparts, and castration also significantly increases ABP-specific UGT activity in the liver. Taken together, our data suggest that androgen increases bladder susceptibility to ABP via liver, likely by modulating an ABP-metabolizing liver enzyme, but exclude UGT as an important mediator.

Total isothiocyanate yield from raw cruciferous vegetables commonly consumed in the United States.

Dietary isothiocyanates are a group of promising chemopreventive agents obtained primarily from cruciferous vegetables. Due to their potent chemopreventive and/or anti-cancer activities, there is a growing interest in assessing dietary isothiocyanate exposure and its impact on human health. Using the HPLC-based cyclocondensation assay, the current study measured total isothiocyanate yield from raw cruciferous vegetables. A total of 73 samples comprising nine types of cruciferous vegetables were analyzed. We observed a wide range of isothiocyanate content across the individual vegetables with an average level of 16.2 µmol/100g wet weight, ranging from 1.5 µmol in raw cauliflower to 61.3 µmol in raw mustard greens. The data represent the maximum amount of isothiocyanates released from the intake of raw cruciferous vegetables. Given that the vegetables assayed in this study include the most commonly consumed cruciferous vegetables in western diets, the data may be particularly useful in estimation of dietary isothiocyanate exposure in these populations. However, due to the variation observed within each vegetable, biomarkers such as urinary isothiocyanate level may be necessary for accurate estimation of individual exposure.

Mechanism of chemical activation of Nrf2.

NF-E2 related factor-2 (Nrf2) promotes the transcription of many cytoprotective genes and is a major drug target for prevention of cancer and other diseases. Indeed, the cancer-preventive activities of several well-known chemical agents were shown to depend on Nrf2 activation. It is well known that chemopreventive Nrf2 activators stabilize Nrf2 by blocking its ubiquitination, but previous studies have indicated that this process occurs exclusively in the cytoplasm. Kelch-like ECH-associated protein 1 (Keap1) binds to Nrf2 and orchestrates Nrf2 ubiquitination, and it has been a widely-held view that inhibition of Nrf2 ubiquitination by chemopreventive agents results from the dissociation of Nrf2 from its repressor Keap1. Here, we show that while the activation of Nrf2 by prototypical chemical activators, including 5,6-dihydrocyclopenta-1,2-dithiole-3-thione (CPDT) and sulforaphane (SF), results solely from inhibition of its ubiquitination, such inhibition occurs predominantly in the nucleus. Moreover, the Nrf2 activators promote Nrf2 association with Keap1, rather than disassociation, which appears to result from inhibition of Nrf2 phosphorylation at Ser40. Available evidence suggests the Nrf2 activators may block Nrf2 ubiquitination by altering Keap1 conformation via reaction with the thiols of specific Keap1 cysteines. We further show that while the inhibitory effects of CPDT and SF on Nrf2 ubiquitination depend entirely on Keap1, Nrf2 is also degraded by a Keap1-independent mechanism. These findings provide significant new insight about Nrf2 activation and suggest that exogenous chemical activators of Nrf2 enter the nucleus to exert most of their inhibitory impact on Nrf2 ubiquitination and degradation.

Identification of an unintended consequence of Nrf2-directed cytoprotection against a key tobacco carcinogen plus a counteracting chemopreventive intervention.

NF-E2-related factor 2 (Nrf2) is a major cytoprotective gene and is a key chemopreventive target against cancer and other diseases. Here we show that Nrf2 faces a dilemma in defense against 4-aminobiphenyl (ABP), a major human bladder carcinogen from tobacco smoke and other environmental sources. Although Nrf2 protected mouse liver against ABP (which is metabolically activated in liver), the bladder level of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP), the predominant ABP-DNA adduct formed in bladder cells and tissues, was markedly higher in Nrf2(+/+) mice than in Nrf2(-/-) mice after ABP exposure. Notably, Nrf2 protected bladder cells against ABP in vitro. Mechanistic investigations showed that the dichotomous effects of Nrf2 could be explained at least partly by upregulation of UDP-glucuronosyltransferase (UGT). Nrf2 promoted conjugation of ABP with glucuronic acid in the liver, increasing urinary excretion of the conjugate. Although glucuronidation of ABP and its metabolites is a detoxification process, these conjugates, which are excreted in urine, are known to be unstable in acidic urine, leading to delivery of the parent compounds to bladder. Hence, although higher liver UGT activity may protect the liver against ABP, it increases bladder exposure to ABP. These findings raise concerns of potential bladder toxicity when Nrf2-activating chemopreventive agents are used in humans exposed to ABP, especially in smokers. We further show that 5,6-dihydrocyclopenta[c][1,2]-dithiole-3(4H)-thione (CPDT) significantly inhibits dG-C8-ABP formation in bladder cells and tissues but does not seem to significantly modulate ABP-catalyzing UGT in liver. Thus, CPDT exemplifies a counteracting solution to the dilemma posed by Nrf2.

Allyl isothiocyanate-rich mustard seed powder inhibits bladder cancer growth and muscle invasion.

Allyl isothiocyanate (AITC), which occurs in many common cruciferous vegetables, was recently shown to be selectively delivered to bladder cancer tissues through urinary excretion and to inhibit bladder cancer development in rats. The present investigation was designed to test the hypothesis that AITC-containing cruciferous vegetables also inhibit bladder cancer development. We focused on an AITC-rich mustard seed powder (MSP-1). AITC was stably stored as its glucosinolate precursor (sinigrin) in MSP-1. Upon addition of water, however, sinigrin was readily hydrolyzed by the accompanying endogenous myrosinase. This myrosinase was also required for full conversion of sinigrin to AITC in vivo, but the matrix of MSP-1 had no effect on AITC bioavailability. Sinigrin itself was not bioactive, whereas hydrated MSP-1 caused apoptosis and G(2)/M phase arrest in bladder cancer cell lines in vitro. Comparison between hydrated MSP-1 and pure sinigrin with added myrosinase suggested that the anticancer effect of MSP-1 was derived principally, if not entirely, from the AITC generated from sinigrin. In an orthotopic rat bladder cancer model, oral MSP-1 at 71.5 mg/kg (sinigrin dose of 9 µmol/kg) inhibited bladder cancer growth by 34.5% (P < 0.05) and blocked muscle invasion by 100%. Moreover, the anticancer activity was associated with significant modulation of key cancer therapeutic targets, including vascular endothelial growth factor, cyclin B1 and caspase 3. On an equimolar basis, the anticancer activity of AITC delivered as MSP-1 appears to be more robust than that of pure AITC. MSP-1 is thus an attractive delivery vehicle for AITC and it strongly inhibits bladder cancer development and progression.

Sulforaphane inhibits 4-aminobiphenyl-induced DNA damage in bladder cells and tissues.

Sulforaphane (SF) is a well-known chemopreventive phytochemical and occurs in broccoli and to a lesser extent in other cruciferous vegetables, whereas 4-aminobiphenyl (ABP) is a major human bladder carcinogen and is present at significant levels in tobacco smoke. Here, we show that SF inhibits ABP-induced DNA damage in both human bladder cells in vitro and mouse bladder tissue in vivo, using dG-C8-ABP as a biomarker, which is the predominant ABP-DNA adduct formed in human bladder cells and tissues. SF activates NF-E2 related factor-2 (Nrf2), which is a well-recognized chemopreventive target and activates the Nrf2-regulated cytoprotective signaling pathway. Comparison between wild-type mice and mice without Nrf2 shows that Nrf2 activation is required by SF for inhibition of ABP-induced DNA damage. Moreover, Nrf2 activation by SF in the bladder occurs primarily in the epithelium, which is the principal site of bladder cancer development. These data, together with our recent observation that SF-enriched broccoli sprout extracts strongly inhibits N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer development, suggest that SF is a highly promising agent for bladder cancer prevention and provides a mechanistic insight into the repeated epidemiological observation that consumption of broccoli is inversely associated with bladder cancer risk and mortality.

Synthesis, biological evaluation, and structure-activity relationships of dithiolethiones as inducers of cytoprotective phase 2 enzymes.

Dithiolethiones are a family of promising cancer chemopreventive agents, and induction of phase 2 enzymes is key to their chemopreventive activities. Two dithiolethiones have been evaluated in humans for cancer prevention. While some chemopreventive activities were detected in several human studies, potential side effects are a concern. Herein, we report structure-activity relationships of 25 dithiolethiones. Several compounds show exceedingly potent and bladder specific activity in phase 2 enzyme induction. Structural features responsible for such activity, as well as those inhibiting the activity, are discussed. Moreover, the compounds activate and depend on Nrf2 for their inductive activities. Nrf2 is a major transcriptional stimulator of cytoprotective genes and is critical for cancer prevention. Thus, several new dithiolethiones that are highly promising for bladder cancer prevention have been identified. Because the compounds act specifically in the bladder, the likelihood of potential systemic toxicity may be low.

Inhibition of bladder cancer development by allyl isothiocyanate.

Bladder cancer is one of the common human cancers and also has a very high recurrence rate. There is a great need for agents capable of inhibiting bladder cancer development and recurrence. Here, we report that allyl isothiocyanate (AITC), an ingredient of many common cruciferous vegetables, potently inhibited the proliferation of bladder carcinoma cell lines in vitro [half maximal inhibitory concentration (IC(50)) of 2.7-3.3 microM], which was associated with profound G(2)/M arrest and apoptosis. In contrast, AITC was markedly less toxic to normal human bladder epithelial cells (IC(50) of 69.4 microM). AITC was then evaluated in two rat bladder cancer models in vivo (an orthotopic model and a subcutaneous model). The orthotopic model closely mimics human bladder cancer development and recurrence. We show that a low oral dose of AITC (1 mg/kg) significantly inhibited the development and muscle invasion of the orthotopic bladder cancers but was ineffective against the subcutaneous xenografts of the same cancer cells in the same animals. This differential effect was explained by our finding that urinary levels of AITC equivalent were two to three orders of magnitude higher than that in the plasma and that its levels in the orthotopic cancer tissues were also three orders of magnitude higher than that in the subcutaneous cancer tissues. Moreover, we show that AITC is a multi-targeted agent against bladder cancer. In conclusion, AITC is selectively delivered to bladder cancer tissue through urinary excretion and potently inhibits bladder cancer development and invasion.

An improved liquid chromatography-tandem mass spectrometry method for the quantification of 4-aminobiphenyl DNA adducts in urinary bladder cells and tissues.

Exposure to 4-aminobiphenyl (4-ABP), an environmental and tobacco smoke carcinogen that targets the bladder urothelium, leads to DNA adduct formation and cancer development [1]. Two major analytical challenges in DNA adduct analysis of human samples have been limited sample availability and the need to reach detection limits approaching the part-per-billion threshold. By operating at nano-flow rates and incorporating a capillary analytical column in addition to an online sample enrichment step, we have developed a sensitive and quantitative HPLC-MS/MS method appropriate for the analysis of such samples. This assay for the deoxyguanosine adduct of 4-ABP (dG-C8-4-ABP) gave mass detection limits of 20amol in 1.25microg of DNA (5 adducts in 10(9) nucleosides) with a linear range of 70amol to 70fmol. 4-ABP-exposed human bladder cells and rat bladder tissue were analyzed in triplicate, and higher dose concentrations led to increased numbers of detected adducts. It was subsequently established that sample requirements could be further reduced to 1microg digestions and the equivalent of 250ng DNA per injection for the detection of low levels of dG-C8-4-ABP in a matrix of exfoliated human urothelial cell DNA. This method is appropriate for the characterization and quantification of DNA adducts in human samples and can lead to a greater understanding of their role in carcinogenesis and also facilitate evaluation of chemopreventive agents.

5,6-Dihydrocyclopenta[c][1,2]-dithiole-3(4H)-thione is a promising cancer chemopreventive agent in the urinary bladder.

It has been widely recognized that induction of Phase 2 enzymes is an effective and sufficient strategy for achieving protection against carcinogenesis. Nrf2 is the unifying master regulator of these enzymes and its activation in various tissues, including the urinary bladder, is associated with inhibition of carcinogenesis. 5,6-Dihydrocyclopenta[c][1,2]-dithiole-3(4H)-thione (CPDT) is a highly potent inducer of Phase 2 enzymes and an activator of Nrf2. In vivo, it is particularly effective in the bladder, showing significant effects in this tissue when dosed to rats at levels as low as 0.98 micromol/(kgday) (0.17 mg/(kg day)). The activities of key Phase 2 enzymes, including glutathione S-transferase, NAD(P)H:quinone:oxidoreductase 1 and glutamate cysteine synthetase, and levels of glutathione were elevated by CPDT in rat bladder in vivo and in cultured bladder cells in vitro. In the bladder, enzyme induction and Nrf2 activation appear to occur exclusively in the epithelium. This is highly significant, since almost all bladder cancers develop from the epithelium. Studies in cultured bladder cells using siRNA to knock down Nrf2 or in cells with total Nrf2 knockout showed that the ability of CPDT to induce Phase 2 enzymes depends completely on Nrf2. In conclusion, CPDT potently and preferentially induces Phase 2 enzymes in the bladder epithelium and Nrf2 is its key mediator.

Structure-activity relationships and organ specificity in the induction of GST and NQO1 by alkyl-aryl isothiocyanates.

PURPOSE: To compare the ability of alkyl-aryl isothiocyanates (ITCs) to increase the activities of the Phase 2 detoxification enzymes NAD[P]H:quinone acceptor oxidoreductase 1 (NQO1) and glutathione S-transferases (GST) in rat tissues in vivo and in cells in vitro. MATERIALS AND METHODS: Twelve alkyl-aryl ITCs and the fully-reduced derivative of benzyl ITC (cyclohexylmethyl ITC) were administered to rats each day for 5 days. The animals were then killed and organs harvested. The ITCs were also evaluated in a bladder cell line in culture. The activities of NQO1 and GST in the organs and cells were measured. RESULTS: In vivo, the organ most susceptible to the inductive activity of the ITCs was the urinary bladder, with alpha-methylbenzyl ITC and cyclohexylmethyl ITC being the most effective. Inductive activity in the bladder in vivo did not, however, correlate with that in bladder cells in vitro. CONCLUSIONS: Induction of Phase 2 enzymes increases resistance to chemical carcinogenesis. ITCs could therefore be valuable chemopreventative agents, and the specificity of these substances toward the urinary bladder suggest that they could be particularly useful for protecting against bladder cancer. In this regard, alpha-methylbenzyl ITC and cyclohexylmethyl ITC could be especially valuable.

Inhibition of urinary bladder carcinogenesis by broccoli sprouts.

Isothiocyanates are a well-known class of cancer chemopreventive agents, and broccoli sprouts are a rich source of several isothiocyanates. We report herein that dietary administration to rats of a freeze-dried aqueous extract of broccoli sprouts significantly and dose-dependently inhibited bladder cancer development induced by N-butyl-N-(4-hydroxybutyl) nitrosamine. The incidence, multiplicity, size, and progression of bladder cancer were all inhibited by the extract, while the extract itself caused no histologic changes in the bladder. Moreover, inhibition of bladder carcinogenesis by the extract was associated with significant induction of glutathione S-transferase and NAD(P)H:quinone oxidoreductase 1 in the bladder, enzymes that are important protectants against oxidants and carcinogens. Isothiocyanates are metabolized to dithiocarbamates in vivo, but dithiocarbamates readily dissociate to isothiocyanates. We found that >70% of the isothiocyanates present in the extract were excreted in the urine as isothiocyanate equivalents (isothiocyanates + dithiocarbamates) in 12 h after a single p.o. dose, indicating high bioavailability and rapid urinary excretion. In addition, the concentrations of isothiocyanate equivalents in the urine of extract-treated rats were 2 to 3 orders of magnitude higher than those in plasma, indicating that the bladder epithelium, the major site of bladder cancer development, is most exposed to p.o. dosed isothiocyanate. Indeed, tissue levels of isothiocyanate equivalents in the bladder were significantly higher than in the liver. In conclusion, broccoli sprout extract is a highly promising substance for bladder cancer prevention and the isothiocyanates in the extract are selectively delivered to the bladder epithelium through urinary excretion.