Edward Y. Kim

A metagenomic analysis of pandemic influenza A (2009 H1N1) infection in patients from North America.

Although metagenomics has been previously employed for pathogen discovery, its cost and complexity have prevented its use as a practical front-line diagnostic for unknown infectious diseases. Here we demonstrate the utility of two metagenomics-based strategies, a pan-viral microarray (Virochip) and deep sequencing, for the identification and characterization of 2009 pandemic H1N1 influenza A virus. Using nasopharyngeal swabs collected during the earliest stages of the pandemic in Mexico, Canada, and the United States (n = 17), the Virochip was able to detect a novel virus most closely related to swine influenza viruses without a priori information. Deep sequencing yielded reads corresponding to 2009 H1N1 influenza in each sample (percentage of aligned sequences corresponding to 2009 H1N1 ranging from 0.0011% to 10.9%), with up to 97% coverage of the influenza genome in one sample. Detection of 2009 H1N1 by deep sequencing was possible even at titers near the limits of detection for specific RT-PCR, and the percentage of sequence reads was linearly correlated with virus titer. Deep sequencing also provided insights into the upper respiratory microbiota and host gene expression in response to 2009 H1N1 infection. An unbiased analysis combining sequence data from all 17 outbreak samples revealed that 90% of the 2009 H1N1 genome could be assembled de novo without the use of any reference sequence, including assembly of several near full-length genomic segments. These results indicate that a streamlined metagenomics detection strategy can potentially replace the multiple conventional diagnostic tests required to investigate an outbreak of a novel pathogen, and provide a blueprint for comprehensive diagnosis of unexplained acute illnesses or outbreaks in clinical and public health settings.

TNF Type 2 Receptor (p75) Lowers the Threshold of T Cell Activation

T cell activation requires a threshold amount of TCR-mediated signals, an amount that is reduced by signals mediated through costimulatory molecules expressed on the T cell surface. Here the role of TNFR2 (p75) as a putative costimulatory receptor for T cell activation was examined. It was found that p75 deficiency in CD8+ T cells increased the requirements for TCR agonist approximately 5-fold. Furthermore, p75−/− T cells display a marked reduction in the proliferative response to TCR agonist. This hypoproliferative response was associated with delayed kinetics of induction of the acute activation markers CD25 and CD69 as well as a marked decrease in the production of IL-2 and IFN-γ. The net result is that very few cells are recruited into the dividing population. Interestingly, CD28 costimulation was only partially effective in rescuing the proliferative defect of p75−/−CD8+ T cells. Thus, p75 provides an important costimulatory signal in addition to that provided by CD28 toward optimal T cell proliferation.

TNF Receptor Type 2 (p75) Functions as a Costimulator for Antigen-Driven T Cell Responses In Vivo

Naive T cells require costimulation for robust Ag-driven differentiation and survival. Members of the TNFR family have been shown to provide costimulatory signals conferring survival at distinct phases of the T cell response. In this study, we show that CD4 and CD8 T cells depend on TNFR type 2 (p75) for survival during clonal expansion, allowing larger accumulation of effector cells and conferring protection from apoptosis for a robust memory pool in vivo. We demonstrate using the MHC class I-restricted 2C TCR and MHC class II-restricted AND TCR transgenic systems that TNFR2 regulates the threshold for clonal expansion of CD4 and CD8 T cell subsets in response to cognate Ag. Using a novel recombinant Listeria monocytogenes (rLM) expressing a secreted form of the 2C agonist peptide (SIY) to investigate the role of TNFR2 for T cell immunity in vivo, we found that TNFR2 controls the survival and accumulation of effector cells during the primary response. TNFR2−/− CD8 T cells exhibit loss of protection from apoptosis that is correlated with diminished survivin and Bcl-2 expression. Null mutant mice were more susceptible to rLM-SIY challenge at high doses of primary infection, correlating with impaired LM-specific T cell response in the absence of TNFR2-mediated costimulation. Moreover, the resulting memory pools specific for SIY and listeriolysin O epitopes derived from rLM-SIY were diminished in TNFR2−/− mice. Thus, examination of Ag-driven T cell responses revealed a hitherto unknown costimulatory function for TNFR2 in regulating T cell survival during the differentiation program elicited by intracellular pathogen in vivo.

Critical Role of TNF Receptor Type-2 (p75) as a Costimulator for IL-2 Induction and T Cell Survival: A Functional Link to CD28

CD28 provides important signals that lower the threshold of T cell activation, augment the production of IL-2, and promote T cell survival. The recent identification of a second family of costimulatory molecules within the TNFR family has reshaped the “two-signal” model of T cell activation. In this study the role of p75 as a T cell costimulatory molecule in controlling cell fate during TCR/CD28-mediated stimulation was examined. We found that p75-deficient T cells possess a profound defect in IL-2 production in response to TCR/CD28-mediated stimulation. Examination of key signaling intermediates revealed that TCR proximal events such as global tyrosine phosphorylation and ZAP70 phosphorylation, as well as downstream MAPK cascades are unperturbed in p75-deficient T cells. In contrast, p75 is nonredundantly coupled to sustained AKT activity and NF-κB activation in response to TCR/CD28-mediated stimulation. Moreover, p75-deficient T cells possess a defect in survival during the early phase of T cell activation that is correlated with a striking defect in Bcl-xL expression. These data indicate discrete effects of p75 on the intracellular signaling milieu during T cell activation, and reveal the synergistic requirement of TCR, CD28, and p75 toward optimal IL-2 induction and T cell survival. We propose that p75 acts as one of the earliest of the identified costimulatory members of the TNFR family, and is functionally linked to CD28 for initiating and determining T cell fate during activation.

Prospective analysis of fluoroscopy duration during ERCP: critical determinants

BACKGROUND Fluoroscopy during ERCP has a linear relationship with radiation, carrying risk of exposure. OBJECTIVE To determine patient, physician, and procedural factors affecting fluoroscopy duration. DESIGN Prospective analysis of ERCPs with evaluation of patient, physician, and procedural variables. SETTING Two tertiary-care hospitals. PATIENTS Consecutive patients undergoing ERCP. INTERVENTIONS ERCP. MAIN OUTCOME MEASUREMENTS Variables associated with prolonged fluoroscopy duration. RESULTS Mean fluoroscopy time (388 ERCPs) was 6.77 minutes (95% CI, 6.15-7.39). No patient factors were found to significantly affect fluoroscopy duration. Fluoroscopy duration was significantly lower for 2 endoscopists compared with the reference endoscopist (average of 4.16 minutes less; 95% CI, -5.48 to -2.48). Multivariable analysis identified variables associated with longer fluoroscopy duration; stent insertion (+3.11 minutes; 95% CI, 1.91-4.30), lithotripsy (+5.74 minutes; 95% CI, 0.931-10.5), needle-knife sphincterotomy (+4.44 minutes; 95% CI, 2.20-6.67), biopsies (+2.11 minutes; 95% CI, 0.025-4.18), use of a guidewire (+1.55 minutes; 95% CI, 0.025-3.07), additional guidewires (+5.61 minutes; 95% CI, 2.69-8.51), and balloon catheter (+4.27 minutes; 95% CI, 3.00-5.53). Mean fluoroscopy duration when a gastroenterology fellow was involved (n = 318) was 7.05 minutes (95% CI, 6.35-7.76) compared with 5.44 minutes (95% CI, 4.26-6.63) when no fellow present (n = 70) (P < .0451). LIMITATIONS Only 2 centers; others may have different results. Not blinded; investigators may change their practice because fluoroscopy was duration studied. Irrelevance of measuring fluoroscopy duration because endoscopists using protection may not have increased radiation exposure. CONCLUSIONS In this prospective analysis, factors associated with fluoroscopy duration included endoscopists; stent insertion; lithotripsy; biopsies; use of a needle-knife, guidewire, and balloon catheter; and involvement of a gastroenterology fellow. These identified variables may help endoscopists predict which procedures are associated with prolonged fluoroscopy duration and may lead to appropriate precautions.

A randomized controlled trial assessing the effect of prescribed patient position changes during colonoscope withdrawal on adenoma detection

BACKGROUND High-quality colonoscope withdrawal technique is associated with a higher adenoma detection rate. Position change is routinely used in barium enema and CT colonography to facilitate adequate distension of the colon and promote movement of fluid from the segment of the colon being assessed. OBJECTIVE To determine whether prescribed position changes during colonoscope withdrawal affect the adenoma detection rate compared with the usual care per endoscopist. DESIGN Prospective, randomized, controlled trial. SETTING Tertiary-care, university-affiliated hospital. PATIENTS Patients referred for outpatient colonoscopy between July 2011 and July 2012 were evaluated for eligibility. Inclusion criteria were outpatient status and age ≥40 years. Exclusion criteria were (1) complete colonoscopy within 1 year before the procedure, (2) inability to provide informed consent, (3) incomplete colonoscopy to the cecum, (4) previous bowel resection, (5) inflammatory bowel disease, (6) colonic polyposis syndrome, (7) inadequate bowel preparation, and (8) musculoskeletal disorder or other mobility issues limiting effective patient position changes during colonoscopy. INTERVENTIONS Prescribed position changes during colonoscope withdrawal. MAIN OUTCOME MEASUREMENTS Polyp detection rate (PDR) and adenoma detection rate (ADR). RESULTS A total of 776 patients were enrolled, with 388 in the dynamic group. There was no difference in PDR (odds ratio [OR] 0.99; P = .93) or ADR (OR 1.17; P = .28). Colonoscope withdrawal time was longer in the dynamic group (median time 466.5 vs 422.5 seconds; P < .0001). LIMITATIONS Single-center study. Indication for procedure not controlled. Lack of standardized bowel preparation and blinding. CONCLUSION Prescribed position changes during colonoscope withdrawal do not affect polyp/adenoma detection compared with the usual practice when the baseline ADR is above the recommended standard. ( CLINICAL TRIAL REGISTRATION NUMBER NCT01395173.).

TNFR2-Deficient Memory CD8 T cells Provide Superior Protection Against Tumor Cell Growth

TNF receptor-2 (TNFR2) plays a critical role in promoting the activation and survival of naive T cells during the primary response. Interestingly, anti-CD3 plus IL-2 activated TNFR2−/− CD8 T cells are highly resistant to activation-induced cell death (AICD), which correlates with high expression levels of prosurvival molecules such as Bcl-2, survivin, and CD127 (IL-7Rα). We determined whether the resistance of activated TNFR2−/− CD8 T cells to AICD contributes to more effective protection against tumor cell growth. We found that during a primary tumor challenge, despite initial inferiority in controlling tumor cell growth, TNFR2−/− mice were able to more effectively control tumor burden over time compared with wild-type (WT) mice. Furthermore, vaccination of TNFR2−/− mice with recombinant Listeria monocytogenes that express OVA confers better protection against the growth of OVA-expressing E.G7 tumor cells relative to similarly vaccinated WT mice. The enhanced protection against tumor cell growth was not due to more effective activation of OVA-specific memory CD8 T cells in vaccinated TNFR2−/− mice. In vitro studies indicate that optimally activated OVA-specific TNFR2−/− CD8 T cells proliferated to the same extent and possess similar cytotoxicity against E.G7 tumor cells as WT CD8 T cells. However, relative to WT cells, activated OVA-specific TNFR2−/− CD8 T cells were highly resistant to AICD. Thus, the enhanced protection against E.G7 in TNFR2−/− mice is likely due to the recruitment and activation of OVA-specific memory TNFR2−/− CD8 T cells and their prolonged survival at the tumor site.

Under “real world” conditions, desflurane increases drug cost without speeding discharge after short ambulatory anesthesia compared to isoflurane

PurposeTo compare the measured “real world” perioperative drug cost and recovery associated with desflurane- and isoflurane-based anesthesia in short (less than one hour) ambulatory surgery.MethodsWe conducted a prospective, randomized, blinded trial with patients undergoing arthroscopic meniscectomy under general anesthesia. Followingiv induction, patients received either isoflurane (group I;n = 25) or desflurane (group D;n = 20) for maintenance. The primary outcome variable was total perioperative drug cost per patient in Canadian dollars. Secondary outcome variables included volatile agent consumption and cost, adjuvant anesthetic and postanesthesia care unit (PACU) drug cost, readiness for PACU discharge, and incidence of adverse events.ResultsTotal perioperative drug cost per patient was

Clinical and endoscopic significance of bowel-wall thickening reported on abdominal computed tomographies in symptomatic patients with no history of gastrointestinal disease.

14.58 ± 6.83 (mean ± standard deviation) for group I, and

Pneumatosis intestinalis after colonoscopy in a crohn's disease patient with mucosal healing

21.47 ± 5.18 for group D (P < 0.001). Isoflurane consumption per patient was 6.0 ± 3.0 mL compared to 18.6 ± 7.7 mL for desflurane (P < 0.0001); corresponding costs were