Katherine Yang

Serotonin regulates pancreatic beta cell mass during pregnancy

During pregnancy, the energy requirements of the fetus impose changes in maternal metabolism. Increasing insulin resistance in the mother maintains nutrient flow to the growing fetus, whereas prolactin and placental lactogen counterbalance this resistance and prevent maternal hyperglycemia by driving expansion of the maternal population of insulin-producing beta cells. However, the exact mechanisms by which the lactogenic hormones drive beta cell expansion remain uncertain. Here we show that serotonin acts downstream of lactogen signaling to stimulate beta cell proliferation. Expression of serotonin synthetic enzyme tryptophan hydroxylase-1 (Tph1) and serotonin production rose sharply in beta cells during pregnancy or after treatment with lactogens in vitro. Inhibition of serotonin synthesis by dietary tryptophan restriction or Tph inhibition blocked beta cell expansion and induced glucose intolerance in pregnant mice without affecting insulin sensitivity. Expression of the Gαq-linked serotonin receptor 5-hydroxytryptamine receptor-2b (Htr2b) in maternal islets increased during pregnancy and normalized just before parturition, whereas expression of the Gαi-linked receptor Htr1d increased at the end of pregnancy and postpartum. Blocking Htr2b signaling in pregnant mice also blocked beta cell expansion and caused glucose intolerance. These studies reveal an integrated signaling pathway linking beta cell mass to anticipated insulin need during pregnancy. Modulators of this pathway, including medications and diet, may affect the risk of gestational diabetes.

Sox9 coordinates a transcriptional network in pancreatic progenitor cells

During pancreas development, both the exocrine and endocrine lineages differentiate from a common pool of progenitor cells with similarities to mature pancreatic duct cells. A small set of transcription factors, including Tcf2, Onecut1, and Foxa2, has been identified in these pancreatic progenitor cells. The Sry/HMG box transcription factor Sox9 is also expressed in the early pancreatic epithelium and is required for normal pancreatic exocrine and endocrine development in humans. In this study, we found Sox9 in mice specifically expressed with the other progenitor transcription factors in both pancreatic progenitor cells and duct cells in the adult pancreas. Sox9 directly bound to all three genes in vitro and in intact cells, and regulated their expression. In turn, both Foxa2 and Tcf2 regulated Sox9 expression, demonstrating feedback circuits between these genes. Furthermore, Sox9 activated the expression of the proendocrine factor Neurogenin3, which also depends on the other members of the progenitor transcription network. These studies indicate that Sox9 plays a dual role in pancreatic progenitor cells: both maintaining a stable transcriptional network and supporting the programs by which these cells differentiate into distinct lineages.

Increased mortality of ventilated patients with endotracheal Pseudomonas aeruginosa without clinical signs of infection.

Objective:To investigate the frequency and outcomes of ventilated patients with newly acquired large burdens of Pseudomonas aeruginosa and to test the hypothesis that large quantities of bacteria are associated with adverse patient outcomes. Design:A prospective, single-center, observational, cohort study. Setting:Medical-surgical intensive care units in a tertiary care university hospital. Patients:All adult patients requiring ≥48 hrs of mechanical ventilation and identified as having newly acquired P. aeruginosa in their lower respiratory tracts between October 2002 and April 2006. Interventions:None. Measurements and Main Results:Daily surveillance cultures of endotracheal aspirates were performed on patients intubated ≥48 hrs; 69 patients with newly acquired P. aeruginosa were enrolled. Daily P. aeruginosa quantification of endotracheal aspirates was performed; clinical signs of infection were noted. Of 45 patients with high P. aeruginosa burdens (≥1,000,000 colony-forming units/mL in endotracheal aspirates; ≥10,000 colony-forming units/mL in bronchoalveolar-lavage), 17 (37.8%) patients did not meet clinical criteria for ventilator-associated pneumonia and had a statistically significant higher risk of death (adjusted hazard ratio, 37.53; 95% confidence interval, 3.79–371.96; p = 0.002) when compared with the patients who had P. aeruginosa ventilator-associated pneumonia. When excluding the ten patients who had ventilator-associated pneumonia attributed to bacteria other than P. aeruginosa or attributed to multiple bacteria including P. aeruginosa, the risk of death remained statistically significant (adjusted hazard ratio, 23.98; 95% confidence interval: 2.49–230.53; p = 0.006). Furthermore, more patients with high P. aeruginosa burdens secreted the type III secretion facilitator protein, PcrV (p = 0.01). Conclusions:A group of patients with large burdens of P. aeruginosa who did not meet clinical criteria for ventilator-associated pneumonia had an increased risk of death when compared with patients who had high P. aeruginosa burdens and met ventilator-associated pneumonia criteria. Patients with high P. aeruginosa burden seemed to possess more virulent strains.

Diagnosis and Treatment of Extended-Spectrum and AmpC β-Lactamase—Producing Organisms

Objective: To review the laboratory diagnosis of extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase–producing bacteria and evaluate potential treatment options. Data Sources: A PubMed search, restricted to English-language articles, was conducted (1966–May 2007) using the search terms ESBL, AmpC, diagnosis, detection, carbapenem, ertapenem, fluoroquinolone, cephalosporin, cefepime, tigecycline, and colistin. Additional references were identified through review of bibliographies of identified articles. Study Selection and Data Extraction: All studies that evaluated laboratory methods for the detection of ESBLs and AmpC β-lactamases and/or the treatment of these organisms were reviewed. All articles that were deemed to be clinically pertinent were included and critically evaluated. Data Synthesis: Numerous laboratory techniques are available for the detection of ESBLs. In contrast, laboratory techniques for detection of AmpC β-lactamases are limited, particularly for plasmid-mediated AmpC β-lactamases. Routine microbiologic testing may not detect ESBLs or AmpC β-lactamases. Optimal antibiotic treatment options are derived from limited observational studies and case reports. Randomized clinical trials evaluating appropriate antibiotic treatment options are lacking. In vitro susceptibility does not always correlate with clinical outcomes. The use of imipenem was associated with the lowest incidence of mortality in patients with bacteremia due to ESBL-producing organisms. Conclusions: Laboratory detection of ESBLs for most organisms is possible with Clinical and Laboratory Standards Institute–recommended testing. However, these tests can be associated with both false negative and false positive results, particularly with organisms that harbor both ESBL- and plasmid-mediated AmpC β-lactamases. No established guidelines exist for the detection of AmpC β-lactamases. Imipenem and meropenem are superior to other antibiotics for the treatment of serious infections due to ESBL and AmpC β-lactamase–producing gram-negative bacteria. While in vitro data demonstrate that tigecycline, ertapenem, and colistin might be potential choices, clinical experience is lacking.

Risk factors and risk adjustment for surgical site infections in pediatric cardiothoracic surgery patients

BACKGROUND The complexity of congenital cardiac defects and the aggressive medical management required to support patients through their recovery place children at high risk for surgical site infection (SSI). METHODS We conducted a retrospective review of children undergoing cardiothoracic surgery at a tertiary care referral center between January 1, 2000, and June 30, 2001. Preoperative, intraoperative, and postoperative data were assessed by multivariate analysis. RESULTS Of 726 surgical procedures performed in 626 patients, SSIs occurred after 46 procedures performed in 46 patients (6.3%). Infections were superficial (n = 22; 47.8%), deep tissue (n = 7; 15.2%), or organ space (n = 17; 37.0%), including 5 episodes of mediastinitis. Median time to SSI was 10 days; 36% of the infections were identified after discharge. On multivariate analysis, children with SSIs were more likely to have been <30 days old (odds ratio [OR], 2.9; 95% confidence interval [CI], 1.2-70), to have a perioperative medical device, and to use parenteral nutrition (OR, 3.3; 95% CI, 1.4-7.9). Multiple severity of illness scores, the Risk Adjustment for Congenital Heart Surgery (RACHS-1) category, and longer duration of postoperative antimicrobials were not associated with SSI. CONCLUSION The use of perioperative medical interventions increases the risk of SSI in young children after cardiac surgery. Prolonged postoperative courses of antimicrobials should be avoided in the absence of documented infection.

Multidrug-Resistant Pseudomonas aeruginosa Ventilator-Associated Pneumonia: The Role of Endotracheal Aspirate Surveillance Cultures

BACKGROUND Inappropriate antibacterial treatment of ventilator-associated pneumonia (VAP) due to multidrug-resistant (MDR) pathogens is associated with increased mortality. Endotracheal aspirate (ETA) surveillance cultures potentially identify MDR pathogens, particularly MDR Pseudomonas aeruginosa, resulting in improved selection of therapy in patients who subsequently develop VAP. OBJECTIVE To investigate the role of ETA surveillance cultures in the identification of MDR P. aeruginosa in newly intubated adults who subsequently develop VAP. METHODS Daily ETA surveillance cultures for P. aeruginosa were collected in all adults newly intubated for 48 hours or more. Patients with preexisting lung disease or colonization or infection with P. aeruginosa were excluded. Risk factors and outcomes of patients newly colonized with MDR P. aeruginosa were assessed. RESULTS Seventy-five patients newly colonized with P aeruginosa were identified. Twenty (27%) of these patients were colonized with a P. aeruginosa isolate that was MDR (resistant to >3 classes of antibiotics). Six patients were colonized by an isolate resistant to all tested classes of antibiotics. Forty-five percent of patients colonized with MDR P. aeruginosa subsequently developed VAP. Prior receipt of fluoroquinolones was an independent predictor of colonization with MDR P. aeruginosa (OR 11.82; 95% CI 2.10 to 66.46; p = 0.005). CONCLUSIONS Performance of routine surveillance cultures may aid in the early detection of MDR P. aeruginosa, improving the initiation of early and appropriate antibiotic therapy for patients who subsequently develop VAP.

Novel Role for SGK3 in Glucose Homeostasis Revealed in SGK3/Akt2 Double-Null Mice

The phosphatidylinositol-3-kinase-dependent kinase, Akt2, plays a central role in mediating insulin effects in glucose-metabolizing tissues. Akt2 knockout mice display insulin resistance with a reactive increase in pancreatic islet mass and hyperinsulinemia. The related phosphatidylinositol-3-kinase-dependent kinase, serum- and glucocorticoid-regulated kinase 3 (SGK3), is essential for normal postnatal hair follicle development but plays no apparent role in glucose homeostasis. We report here an unexpected role of SGK3 in islet β-cell function, which is revealed in Akt2/SGK3 double-knockout (DKO) mice. DKO mice have markedly worse glucose homeostasis than Akt2 single-null animals, including greater baseline glucose, and greater rise in blood glucose after glucose challenge. However, surprisingly, our data strongly support the idea that this exacerbation of the glucose-handling defect is due to impaired β-cell function, rather than increased insulin resistance in peripheral tissues. DKO mice had lower plasma insulin and C-peptide levels, lower β-cell mass, reduced glucose-stimulated insulin secretion, and greater sensitivity to exogenous insulin than Akt2 single nulls. We further demonstrated that SGK3 is strongly expressed in normal mouse islets and, interestingly, that β-catenin expression is dramatically lower in the islets of DKO mice than in those of Akt2(-/-)/SGK3(+/+) or Akt2(-/-)/SGK3(+/-) mice. Taken together, these data strongly suggest that SGK3 plays a previously unappreciated role in glucose homeostasis, likely through direct effects within β-cells, to stimulate proliferation and insulin release, at least in part by controlling the expression and activity of β-catenin.

New In Vitro Model To Study the Effect of Human Simulated Antibiotic Concentrations on Bacterial Biofilms

ABSTRACT A new in vitro pharmacokinetic/pharmacodynamic simulator for bacterial biofilms utilizing flow cell technology and confocal laser scanning microscopy is described. The device has the ability to simulate the changing antibiotic concentrations in humans associated with intravenous dosing on bacterial biofilms grown under continuous culture conditions. The free drug concentrations of a single 2-g meropenem intravenous bolus dose and first-order elimination utilizing a half-life of 0.895 h (elimination rate constant, 0.776 h−1) were simulated. The antibacterial activity of meropenem against biofilms of Pseudomonas aeruginosa PAO1 and three clinical strains isolated from patients with cystic fibrosis was investigated. Additionally, the effect of meropenem on PAO1 biofilms cultured for 24 h versus that on biofilms cultured for 72 h was examined. Using confocal laser scanning microscopy, rapid biofilm killing was observed in the first hour of the dosing interval for all biofilms. However, for PAO1 biofilms cultured for 72 h, only bacterial subpopulations at the periphery of the biofilm were affected, with subpopulations at the substratum remaining viable, even at the conclusion of the dosing interval. The described model is a novel method to investigate antimicrobial killing of bacterial biofilms using human simulated concentrations.

Determination of meropenem in bacterial media by LC–MS/MS

To support the development of a dynamic in vitro human pharmacokinetic/pharmacodynamic simulation model for biofilm-mediated infections and study stability of meropenem, an LC-MS/MS method for the determination of meropenem in Luria Bertani (LB) media was developed and validated in an API2000 LC-MS/MS system. A partial validation was also performed in M9 media. Sample aliquots of 100μL (or 25μL for M9 media) were mixed with the internal standard (IS) ceftazidime and filtered. The filtrate was directly injected onto a C8 column eluted with ammonium formate (10mM, pH 4) and acetonitrile (0.1% formic acid) in a gradient mode. ESI(+) and MRM with ion pair m/z 384→68 for meropenem and m/z 547→468 for the IS were used for quantification. The calibration curve concentration range was 50 to 25,000ng/mL. The recovery was over 98%. In LB media, significant signal suppression was observed throughout the time period of detection when compared with mobile phase solvents, but the matrix effect was compensated well with the IS. In M9 media, much less signal suppression was observed. The method is simple, fast, and reliable. Using the method, stability of meropenem in LB and M9 media were tested. No significant degradation was observed for at least 8h in both LB media (37°C) and M9 media (30°C), but more than 15% degradation was observed overnight (∼20h). The method was transferred to an API5000 LC-MS/MS system using meropenem-d6 as the IS.

A Whole-Genome RNA Interference Screen Reveals a Role for Spry2 in Insulin Transcription and the Unfolded Protein Response

Insulin production by the pancreatic β-cell is required for normal glucose homeostasis. While key transcription factors that bind to the insulin promoter are known, relatively little is known about the upstream regulators of insulin transcription. Using a whole-genome RNA interference screen, we uncovered 26 novel regulators of insulin transcription that regulate diverse processes including oxidative phosphorylation, vesicle traffic, and the unfolded protein response (UPR). We focused on Spry2—a gene implicated in human type 2 diabetes by genome-wide association studies but without a clear connection to glucose homeostasis. We showed that Spry2 is a novel UPR target and its upregulation is dependent on PERK. Knockdown of Spry2 resulted in reduced expression of Serca2, reduced endoplasmic reticulum calcium levels, and induction of the UPR. Spry2 deletion in the adult mouse β-cell caused hyperglycemia and hypoinsulinemia. Our study greatly expands the compendium of insulin promoter regulators and demonstrates a novel β-cell link between Spry2 and human diabetes.