Systems-level analysis of innate immunity.

Systems-level analysis of biological processes strives to comprehensively and quantitatively evaluate the interactions between the relevant molecular components over time, thereby enabling development of models that can be employed to ultimately predict behavior. Rapid development in measurement technologies (omics), when combined with the accessible nature of the cellular constituents themselves, is allowing the field of innate immunity to take significant strides toward this lofty goal. In this review, we survey exciting results derived from systems biology analyses of the immune system, ranging from gene regulatory networks to influenza pathogenesis and systems vaccinology.

Lipidomic profiling of influenza infection identifies mediators that induce and resolve inflammation.

Bioactive lipid mediators play a crucial role in the induction and resolution of inflammation. To elucidate their involvement during influenza infection, liquid chromatography/mass spectrometry lipidomic profiling of 141 lipid species was performed on a mouse influenza model using two viruses of significantly different pathogenicity. Infection by the low-pathogenicity strain X31/H3N2 induced a proinflammatory response followed by a distinct anti-inflammatory response; infection by the high-pathogenicity strain PR8/H1N1 resulted in overlapping pro- and anti-inflammatory states. Integration of the large-scale lipid measurements with targeted gene expression data demonstrated that 5-lipoxygenase metabolites correlated with the pathogenic phase of the infection, whereas 12/15-lipoxygenase metabolites were associated with the resolution phase. Hydroxylated linoleic acid, specifically the ratio of 13- to 9-hydroxyoctadecadienoic acid, was identified as a potential biomarker for immune status during an active infection. Importantly, some of the findings from the animal model were recapitulated in studies of human nasopharyngeal lavages obtained during the 2009-2011 influenza seasons.

Mechanisms of gelofusine protection in an in vitro model of polymyxin B-associated renal injury.

Polymyxins are a last-resort treatment option for multidrug-resistant gram-negative bacterial infections, but they are associated with nephrotoxicity. Gelofusine was previously shown to reduce polymyxin-associated kidney injury in an animal model. However, the mechanism(s) of renal protection has not been fully elucidated. Here, we report the use of a cell culture model to provide insights into the mechanisms of renal protection. Murine epithelial proximal tubular cells were exposed to polymyxin B. Cell viability, lactate dehydrogenase (LDH) release, polymyxin B uptake, mitochondrial superoxide production, nuclear morphology, and apoptosis activation were evaluated with or without concomitant gelofusine. A megalin knockout cell line was used as an uptake inhibition control. Methionine was included in selected experiments as an antioxidant control. A polymyxin B concentration-dependent reduction in cell viability was observed. Increased viability was observed in megalin knockout cells following comparable polymyxin B exposures. Compared with polymyxin B exposure alone, concomitant gelofusine significantly increased cell viability as well as reduced LDH release, polymyxin B uptake, mitochondrial superoxide, and apoptosis. Gelofusine and methionine were more effective at reducing renal cell injury in combination than either agent alone. In conclusion, the mechanisms of renal protection by gelofusine involve decreasing cellular drug uptake, reducing subsequent oxidative stress and apoptosis activation. These findings would be valuable for translational research into clinical strategies to attenuate drug-associated acute kidney injury.NEW & NOTEWORTHY Gelofusine is a gelatinous saline solution with the potential to attenuate polymyxin-associated nephrotoxicity. We demonstrated that the mechanisms of gelofusine renal protection involve reducing polymyxin B uptake by proximal tubule cells, limiting subsequent oxidative stress and apoptosis activation. In addition, gelofusine was more effective at reducing cellular injury than a known antioxidant control, methionine, and a megalin knockout cell line, indicating that gelofusine likely has additional pharmacological properties besides only megalin inhibition.

The emergence of cefiderocol resistance in Pseudomonas aeruginosa from a heteroresistant isolate during prolonged therapy.

Cefiderocol is a siderophore cephalosporin designed to target multi-drug-resistant Gram-negative bacteria. Previously, the emergence of cefiderocol non-susceptibility has been associated with mutations in the chromosomal cephalosporinase (PDC) along with mutations in the PirA and PiuA/D TonB-dependent receptor pathways. Here, we report a clinical case of cefiderocol-resistant P. aeruginosa that emerged in a patient during treatment. This resistance was associated with mutations not previously reported, suggesting potential novel pathways to cefiderocol resistance.

Zileuton ameliorates aminoglycoside and polymyxin-associated acute kidney injury in an animal model.

OBJECTIVES: Aminoglycosides and polymyxins are antibiotics with in vitro activity against MDR Gram-negative bacteria. However, their clinical use is hindered by dose-limiting nephrotoxicity. The objective of this project was to determine if zileuton can reduce nephrotoxicity associated with amikacin and polymyxin B in a rat model of acute kidney injury. METHODS: Sprague Dawley rats (n = 10, both genders) were administered either amikacin (300 mg/kg) or polymyxin B (20 mg/kg) daily for 10 days. Zileuton (4 and 10 mg/kg) was delivered intraperitoneally 15 min before antibiotic administration. Blood samples were collected at baseline and daily to determine serum creatinine concentration. Nephrotoxicity was defined as a ≥2× elevation of baseline serum creatinine. Time-to-event analysis and log rank test were used to compare the onset of nephrotoxicity in different cohorts. Histopathological analysis was also conducted to characterize the extent of kidney injury. RESULTS: Animals receiving amikacin or polymyxin B alone had nephrotoxicity rates of 90% and 100%, respectively. The overall rate was reduced to 30% in animals receiving adjuvant zileuton. The onset of nephrotoxicity associated with amikacin and polymyxin B was also significantly delayed by zileuton at 4 and 10 mg/kg, respectively. Histopathology confirmed reduced kidney injury in animals receiving amikacin concomitant with zileuton. CONCLUSIONS: Our pilot data suggest that zileuton has the potential to attenuate nephrotoxicity associated with last-line antibiotics. This would allow these antibiotics to treat MDR Gram-negative bacterial infections optimally without dose-limiting constraints. Further studies are warranted to optimize drug delivery and dosing in humans.

Plain language summary of the HIMALAYA study: tremelimumab and durvalumab for unresectable hepatocellular carcinoma (liver cancer).

WHAT IS THIS SUMMARY ABOUT?: This is a summary of results from a phase 3 clinical study called HIMALAYA. HIMALAYA looked at treatment with one dose of a medication called tremelimumab combined with multiple doses of a medication called durvalumab (the STRIDE regimen) or multiple doses of durvalumab alone. These treatments were compared with a medication called sorafenib in participants with unresectable hepatocellular carcinoma (HCC). HCC is a type of liver cancer that is difficult to treat because it is often diagnosed when it is unresectable, meaning it can no longer be removed with surgery. Sorafenib has been the main treatment for unresectable HCC since 2007. However, people who take sorafenib may experience side effects that can reduce their quality of life, so alternative medicines are being trialed. Tremelimumab and durvalumab are types of drugs called immunotherapies, and they both work in different ways to help the body's immune system fight cancer. WHAT WERE THE RESULTS OF THE STUDY?: Participants who took STRIDE lived longer than participants who took sorafenib, whilst participants who took durvalumab alone lived a similar length of time as participants who took sorafenib. Participants who took STRIDE or durvalumab had a lower relative risk of experiencing worsening in their quality of life than participants who took sorafenib. The side effects that participants who received STRIDE or durvalumab experienced were expected for these types of treatments and could mostly be managed. WHAT DO THE RESULTS OF THE STUDY MEAN?: Overall, STRIDE is more effective than sorafenib for people with unresectable HCC.

Growth of Acinetobacter baumannii impacted by iron chelation.

Acinetobacter baumannii (AB) has become multidrug-resistant (MDR) in recent years, and, currently, there are limited effective treatment options. Nutrient metals (e.g. iron) are essential to the metabolic functions of AB. This study examined the impact of iron chelation on the growth of AB in vitro and in vivo. Susceptible and MDR-AB bloodstream isolates (n = 9) were recovered from different patients between 2011 and 2018. Clonal diversity was ascertained by Fourier-transform infrared spectroscopy. In vitro bacterial densities were measured over 20 h to determine growth profiles. Variable amounts of a chelating agent [deferiprone (DFP)] were added to create a concentration gradient. Galleria mellonella larvae were inoculated with an isolate, with and without DFP. Quantitative culture was used to ascertain the bacterial burden of aggregate larvae immediately and 4 h post-infection. Increasing concentrations of DFP caused a transient and concentration-dependent hindrance to in vitro growth, compared to the no-treatment group. In vivo bacterial burden immediately post-infection in both groups was comparable. After 4 h, the burden was much higher in the control group comparatively (8.7 and 6.7 log CFU g-1). These results support that micro-nutrient limitation has the potential of being a novel approach for treating high-risk infections due to MDR-AB.

PPARα exacerbates necroptosis, leading to increased mortality in postinfluenza bacterial superinfection.

Patients infected with influenza are at high risk of secondary bacterial infection, which is a major proximate cause of morbidity and mortality. We have shown that in mice, prior infection with influenza results in increased inflammation and mortality upon Staphylococcus aureus infection, recapitulating the human disease. Lipidomic profiling of the lungs of superinfected mice revealed an increase in CYP450 metabolites during lethal superinfection. These lipids are endogenous ligands for the nuclear receptor PPARα, and we demonstrate that Ppara-/- mice are less susceptible to superinfection than wild-type mice. PPARα is an inhibitor of NFκB activation, and transcriptional profiling of cells isolated by bronchoalveolar lavage confirmed that influenza infection inhibits NFκB, thereby dampening proinflammatory and prosurvival signals. Furthermore, network analysis indicated an increase in necrotic cell death in the lungs of superinfected mice compared to mice infected with S. aureus alone. Consistent with this, we observed reduced NFκB-mediated inflammation and cell survival signaling in cells isolated from the lungs of superinfected mice. The kinase RIPK3 is required to induce necrotic cell death and is strongly induced in cells isolated from the lungs of superinfected mice compared to mice infected with S. aureus alone. Genetic and pharmacological perturbations demonstrated that PPARα mediates RIPK3-dependent necroptosis and that this pathway plays a central role in mortality following superinfection. Thus, we have identified a molecular circuit in which infection with influenza induces CYP450 metabolites that activate PPARα, leading to increased necrotic cell death in the lung which correlates with the excess mortality observed in superinfection.

Randomised, Phase II study of selumetinib, an oral inhibitor of MEK, in combination with cisplatin and gemcitabine chemotherapy for patients with advanced biliary tract cancer.

INTRODUCTION: Cisplatin and gemcitabine (CisGem) are standard chemotherapy for advanced biliary tract cancer (BTC). The MEK inhibitor selumetinib showed synergy with gemcitabine when administered sequentially in BTC. This randomised Phase 2 trial aimed to assess the efficacy of sequential or continuous selumetinib with CisGem. METHODS: Patients with advanced BTC received CisGem; arm A included selumetinib every day, arm B: selumetinib, days 1-5, 8-19 each cycle. Arm C received CisGem alone. Selumetinib was dosed at 75 mg BID but amended to 50 mg BID due to toxicity. RESULTS: In all, 51 participants were evaluable for response. No significant difference was seen in mean change in tumour size at 10 weeks between arms A and C (-7.8% vs -12.8%, P = 0.54) or arms B and C (-15% vs -12.8%, P = 0.78). There was no difference in median progression-free survival (6.0, 7.0, 6.3 months, P > 0.95) or overall survival (11.7, 11.7, 12.8 months, P = 0.70) for arms A, B and C, respectively. More participants experienced grade 3-4 toxicities in selumetinib-containing arms. More participants in arm A required chemotherapy dose reductions (P = 0.01) with lower chemotherapy dose intensity during the first 10 weeks. CONCLUSION: Adding sequential or continuous selumetinib to CisGem failed to improve efficacy and increased toxicity in patients with advanced BTC.

Case Commentary: Novel Therapy for Multidrug-Resistant Acinetobacter baumannii Infection.

Acinetobacter baumannii is a pathogenic bacterium commonly associated with multidrug resistance. In this issue of Antimicrobial Agents and Chemotherapy, a challenging case of ventilator-associated pneumonia is presented in which bacteriophage therapy was used as a last resort treatment in combination with systemic antibiotics. The data are promising, and several key areas are highlighted for future research.

Optimal ceftazidime/avibactam dosing exposure against KPC-producing Klebsiella pneumoniae.

OBJECTIVES: Infections due to carbapenem-resistant Enterobacterales are considered urgent public health threats and often treated with a ß-lactam/ß-lactamase inhibitor combination. However, clinical treatment failure and resistance emergence have been attributed to inadequate dosing. We used a novel framework to provide insights of optimal dosing exposure of ceftazidime/avibactam. METHODS: Seven clinical isolates of Klebsiella pneumoniae producing different KPC variants were examined. Ceftazidime susceptibility (MIC) was determined by broth dilution using escalating concentrations of avibactam. The observed MICs were characterized as response to avibactam concentrations using an inhibitory sigmoid Emax model. Using the best-fit parameter values, %fT>MICi was estimated for various dosing regimens of ceftazidime/avibactam. A hollow-fibre infection model (HFIM) was subsequently used to ascertain the effectiveness of selected regimens over 120 h. The drug exposure threshold associated with bacterial suppression was identified by recursive partitioning. RESULTS: In all scenarios, ceftazidime MIC reductions were well characterized with increasing avibactam concentrations. In HFIM, bacterial regrowth over time correlated with emergence of resistance. Overall, suppression of bacterial regrowth was associated with %fT>MICi ≥ 76.1% (100% versus 18.2%; P < 0.001). Using our framework, the optimal drug exposure could be achieved with ceftazidime/avibactam 2.5 g every 12 h in 5 out of 7 isolates. Furthermore, ceftazidime/avibactam 2.5 g every 8 h can suppress an isolate deemed resistant based on conventional susceptibility testing method. CONCLUSIONS: An optimal drug exposure to suppress KPC-producing bacteria was identified. The novel framework is informative and may be used to guide optimal dosing of other ß-lactam/ß-lactamase inhibitor combinations. Further in vivo investigations are warranted.

Discerning in vitro pharmacodynamics from OD measurements: A model-based approach.

Time-kill experiments can discern the pharmacodynamics of infectious bacteria exposed to antibiotics in vitro, and thus help guide the design of effective therapies for challenging clinical infections. This task is resource-limited, therefore typically bypassed in favor of empirical shortcuts. The resource limitation could be addressed by continuously assessing the size of a bacterial population under antibiotic exposure using optical density measurements. However, such measurements count both live and dead cells and are therefore unsuitable for declining populations of live cells. To fill this void, we develop here a model-based method that infers the count of live cells in a bacterial population exposed to antibiotics from continuous optical-density measurements of both live and dead cells combined. The method makes no assumptions about the underlying mechanisms that confer resistance and is widely applicable. Use of the method is demonstrated by an experimental study on Acinetobacter baumannii exposed to levofloxacin.

New ß-Lactam-ß-Lactamase Inhibitor Combinations.

The limited armamentarium against drug-resistant Gram-negative bacilli has led to the development of several novel ß-lactam-ß-lactamase inhibitor combinations (BLBLIs). In this review, we summarize their spectrum of in vitro activities, mechanisms of resistance, and pharmacokinetic-pharmacodynamic (PK-PD) characteristics. A summary of available clinical data is provided per drug. Four approved BLBLIs are discussed in detail. All are options for treating multidrug-resistant (MDR) Enterobacterales and Pseudomonas aeruginosa Ceftazidime-avibactam is a potential drug for treating Enterobacterales producing extended-spectrum ß-lactamase (ESBL), Klebsiella pneumoniae carbapenemase (KPC), AmpC, and some class D ß-lactamases (OXA-48) in addition to carbapenem-resistant Pseudomonas aeruginosa Ceftolozane-tazobactam is a treatment option mainly for carbapenem-resistant P. aeruginosa (non-carbapenemase producing), with some activity against ESBL-producing Enterobacterales Meropenem-vaborbactam has emerged as treatment option for Enterobacterales producing ESBL, KPC, or AmpC, with similar activity as meropenem against P. aeruginosa Imipenem-relebactam has documented activity against Enterobacterales producing ESBL, KPC, and AmpC, with the combination having some additional activity against P. aeruginosa relative to imipenem. None of these drugs present in vitro activity against Enterobacterales or P. aeruginosa producing metallo-ß-lactamase (MBL) or against carbapenemase-producing Acinetobacter baumannii Clinical data regarding the use of these drugs to treat MDR bacteria are limited and rely mostly on nonrandomized studies. An overview on eight BLBLIs in development is also provided. These drugs provide various levels of in vitro coverage of carbapenem-resistant Enterobacterales, with several drugs presenting in vitro activity against MBLs (cefepime-zidebactam, aztreonam-avibactam, meropenem-nacubactam, and cefepime-taniborbactam). Among these drugs, some also present in vitro activity against carbapenem-resistant P. aeruginosa (cefepime-zidebactam and cefepime-taniborbactam) and A. baumannii (cefepime-zidebactam and sulbactam-durlobactam).

Optimizing pharmacokinetics/pharmacodynamics of ß-lactam/ß-lactamase inhibitor combinations against high inocula of ESBL-producing bacteria.

OBJECTIVES: Reduced in vitro ß-lactam activity against a dense bacterial population is well recognized. It is commonly attributed to the presence of ß-lactamase(s) and it is unknown whether the inoculum effect could be diminished by a ß-lactamase inhibitor. We evaluated different ß-lactam/ß-lactamase inhibitor combinations in suppressing a high inoculum of ESBL-producing bacteria. METHODS: Three clinical isolates expressing representative ESBLs (CTX-M-15 and SHV-12) were examined. The impact of escalating ß-lactamase inhibitor (tazobactam or avibactam) concentrations on ß-lactam (piperacillin or ceftazidime) MIC reduction was characterized by an inhibitory sigmoid Emax model. The effect of various dosing regimens of ß-lactam/ß-lactamase inhibitor combinations was predicted using %T>MICi and selected exposures were experimentally validated in a hollow-fibre infection model over 120 h. The threshold exposure to suppress bacterial regrowth was identified using recursive partitioning. RESULTS: A concentration-dependent reduction in ß-lactam MIC was observed (r2 ≥0.93). Regrowth could be suppressed in all six experiments using %T>MICi ≥73.6%, but only one out of six experiments below the threshold (P = 0.015). The exposures to suppress regrowth might be attained using the clinical dose of avibactam, but a much higher dose than the standard dose would be needed for tazobactam. CONCLUSIONS: A dense population of ESBL-producing bacteria could be suppressed by an optimized dosing regimen of selected ß-lactam/ß-lactamase inhibitor combinations. The reversibility of enzyme inhibition could play an important role in diminishing the inoculum effect. In vivo investigations to validate these findings are warranted.

SIMULTANEOUS IN VITRO SIMULATION OF MULTIPLE ANTIMICROBIAL AGENTS WITH DIFFERENT ELIMINATION HALF-LIVES IN A PRE-CLINICAL INFECTION MODEL.

Combination therapy for treatment of multi-drug resistant bacterial infections is becoming common. In vitro testing of drug combinations under realistic pharmacokinetic conditions is needed before a corresponding combination is eventually put into clinical use. The current standard for design of such in vitro simulations for drugs with different half-lives is heuristic and limited to two drugs. To address that void, we develop a rigorous design method suitable for an arbitrary number of N drugs with different half-lives. The method developed offers substantial flexibility and produces novel designs even for two drugs. Explicit design equations are rigorously developed and are suitable for immediate use by experimenters. These equations were used in experimental verification using a combination of three antibiotics with distinctly different half-lives. In addition to antibiotics, the method is applicable to any anti-infective or anti-cancer drugs with distinct elimination pharmacokinetics.

Optimizing Pharmacokinetics-Pharmacodynamics of Antimicrobial Management in Patients with Sepsis: A Review.

Critically ill patients with sepsis or septic shock are at an increased risk of death. Early and aggressive interventions are essential for improving clinical outcomes. There are a number of therapeutic and practical challenges in the management of antimicrobials in patients with sepsis. These include the timely selection and administration of appropriate antimicrobials, significant physiological alterations that can influence antimicrobial pharmacokinetics, and significant interpatient variability of antimicrobial concentrations using standard dosing approaches. Understanding the impact of these factors on the probability of attaining pharmacokinetic-pharmacodynamic target goals is essential to guide optimal therapy. Using rapid diagnostic technology could facilitate timely selection of antimicrobials, and therapeutic drug monitoring would provide a more individualized dosing approach. Using an interdisciplinary sepsis team would also be beneficial in coordinating efforts to overcome the challenges encountered during this critical period to ensure optimal care.

Characterization of Amikacin Drug Exposure and Nephrotoxicity in an Animal Model.

Despite excellent in vitro activity, aminoglycosides are used conservatively to treat multidrug-resistant bacterial infections due to their associated nephrotoxicity. Aminoglycosides are known to accumulate in the kidneys, but the quantitative relationship between drug exposures and nephrotoxicity is not well established. To bridge the knowledge gap, the objective of this study was to develop an animal model with clinically relevant conditions to mimic human disease progression. Single-dose pharmacokinetics were studied in Sprague-Dawley rats dosed either with 100 or 500 mg/kg of body weight of amikacin subcutaneously. Serial blood samples were collected, and serum amikacin concentrations were measured using liquid chromatography tandem mass spectrometry. Rats were also dosed with amikacin once daily for up to 10 days; blood samples were taken at baseline and daily to detect nephrotoxicity (defined as doubling of serum creatinine from baseline). Kidneys from both studies were harvested from selected rats, and amikacin concentrations in renal tissues were measured. A dose-dependent increase in systemic area under the curve (AUC) was observed, which ranged from approximately 1/3 (AUC of 53 mg·h/liter) to 3 times (AUC of 650 mg·h/liter) the expected exposure resulting from standard dosing in humans. Nephrotoxicity was significantly higher in rats given 500 mg/kg (100% versus 30%, P = 0.003). Kaplan-Meier analysis also showed a significant difference in nephrotoxicity onset between the two groups (P = 0.001). Finally, analysis of the renal tissues showed that the accumulation of amikacin could be associated with nephrotoxicity. These results are consistent with clinical observations, which support using this model in the future to investigate an intervention(s) that can be used clinically to alleviate nephrotoxicity.

Local Tissue Response to Subcutaneous Administration of Ceftriaxone in an Animal Model.

Subcutaneous administration is a novel way to deliver antibiotics for an infection, but intolerability has been reported. Evaluating the local tolerability of subcutaneously administered antibiotics is not standardized. The goal of this study was to develop an animal model to assess the subcutaneous administration of ceftriaxone. Sprague-Dawley rats were given daily subcutaneous injections for 12 days. The back of each animal was divided into 4 quadrants, with injections rotating each day among the quadrants. Ceftriaxone (1,000 mg/kg of body weight daily) was given in different concentrations and durations. Normal saline and potassium chloride solutions (2 meq/2 ml) were used as negative and positive controls, respectively. After the treatment course, skin samples were biopsied, and the local inflammatory response was assessed histologically using a semiquantitative scoring system. The histopathology scores were compared using a Kruskal-Wallis test. Injections with potassium chloride resulted in full-thickness skin necrosis with subcutaneous atrophy that was not seen in the saline-injected animals; inflammation of the muscular panniculus was observed, with various degrees of myocyte injury. Serosanguinous cavity formation in the subcutaneous compartment was observed when ceftriaxone (125 mg/ml) was given as a bolus injection, but the extent of the local tissue response was remarkably reduced when the same ceftriaxone dose was given at a lower concentration (25 mg/ml) over 120 min (P = 0.63, compared to saline controls). At a low concentration, ceftriaxone infusion was found to be well tolerated in this animal tissue necrosis model. If validated, the model could be an instrumental platform to evaluate different pharmaceutical formulations for subcutaneous delivery.

Oncology education for family medicine residents: a national needs assessment survey.

BACKGROUND: This study aimed to determine the current state of oncology education in Canadian family medicine postgraduate medical education programs (FM PGME) and examine opinions regarding optimal oncology education in these programs. METHODS: A survey was designed to evaluate ideal and current oncology teaching, educational topics, objectives, and competencies in FM PGMEs. The survey was sent to Canadian family medicine (FM) residents and program directors (PDs). RESULTS: In total, 150 residents and 17 PDs affiliated with 16 of 17 Canadian medical schools completed the survey. The majority indicated their programs do not have a mandatory clinical rotation in oncology (79% residents, 88% PDs). Low rates of residents (7%) and PDs (13%) reported FM residents being adequately prepared for their role in caring for cancer patients (p = 0.03). Residents and PDs believed the most optimal method of teaching oncology is through clinical exposure (65% residents, 80% PDs). Residents and PDs agreed the most important topics to learn (rated ≥4.7 on 5-point Likert scale) were: performing pap smears, cancer screening/prevention, breaking bad news, and approach to patient with increased cancer risk. According to residents, other important topics such as appropriate cancer patient referrals, managing cancer complications and post-treatment surveillance were only taught at frequencies of 52, 40 and 36%, respectively. CONCLUSIONS: Current FM PGME oncology education is suboptimal, although the degree differs in the opinion of residents and PDs. This study identified topics and methods of education which could be focussed upon to improve FM oncology education.

Cytotoxic Curli Intermediates Form during Salmonella Biofilm Development.

Enterobacteriaceae produce amyloid proteins called curli that are the major proteinaceous component of biofilms. Amyloids are also produced by humans and are associated with diseases such as Alzheimer's. During the multistep process of amyloid formation, monomeric subunits form oligomers, protofibrils, and finally mature fibrils. Amyloid ß oligomers are more cytotoxic to cells than the mature amyloid fibrils. Oligomeric intermediates of curli had not been previously detected. We determined that turbulence inhibited biofilm formation and that, intriguingly, curli aggregates purified from cultures grown under high-turbulence conditions were structurally smaller and contained less DNA than curli preparations from cultures grown with less turbulence. Using flow cytometry analysis, we demonstrated that CsgA was expressed in cultures exposed to higher turbulence but that these cultures had lower levels of cell death than less-turbulent cultures. Our data suggest that the DNA released during cell death drives the formation of larger fibrillar structures. Consistent with this idea, addition of exogenous genomic DNA increased the size of the curli intermediates and led to binding to thioflavin T at levels observed with mature aggregates. Similar to the intermediate oligomers of amyloid ß, intermediate curli aggregates were more cytotoxic than the mature curli fibrils when incubated with bone marrow-derived macrophages. The discovery of cytotoxic curli intermediates will enable research into the roles of amyloid intermediates in the pathogenesis of Salmonella and other bacteria that cause enteric infections.IMPORTANCE Amyloid proteins are the major proteinaceous components of biofilms, which are associated with up to 65% of human bacterial infections. Amyloids produced by human cells are also associated with diseases such as Alzheimer's. The amyloid monomeric subunits self-associate to form oligomers, protofibrils, and finally mature fibrils. Amyloid ß oligomers are more cytotoxic to cells than the mature amyloid fibrils. Here we detected oligomeric intermediates of curli for the first time. Like the oligomers of amyloid ß, intermediate curli fibrils were more cytotoxic than the mature curli fibrillar aggregates when incubated with bone marrow-derived macrophages. The discovery of cytotoxic curli intermediates will enable research into the roles of amyloid intermediates in the pathogenesis of Salmonella and other bacteria that cause enteric infections.