Effectiveness of a Multifaceted Implementation Strategy to Increase Equitable Hospital at Home Utilization: An Interrupted Time Series Analysis.

BACKGROUND: The number of Hospital-at-Home (HaH) programs rapidly increased during the COVID-19 pandemic and after issuance of Centers for Medicare and Medicaid Services' (CMS) Acute Hospital Care at Home (AHCaH) waiver. However, there remains little evidence on effective strategies to equitably expand HaH utilization. OBJECTIVE: Evaluate the effects of a multifaceted implementation strategy on HaH utilization over time. DESIGN: Before and after implementation evaluation using electronic health record (EHR) data and interrupted time series analysis, complemented by qualitative interviews with key stakeholders. PARTICIPANTS: Between December 2021 and December 2022, we identified adults hospitalized at six hospitals in North Carolina approved by CMS to participate in the AHCaH waiver program. Eligible adults met criteria for HaH transfer (HaH-eligible clinical condition, qualifying home environment). We conducted semi-structured interviews with 12 HaH patients and 10 referring clinicians. INTERVENTIONS: Two strategies were studied. The discrete implementation strategy (weeks 1-12) included clinician-directed educational outreach. The multifaceted implementation strategy (weeks 13-54) included ongoing clinician-directed educational outreach, local HaH assistance via nurse navigators, involvement of clinical service line executives, and individualized audit and feedback. MEASURES: We assessed weekly averaged HaH capacity utilization, weekly counts of unique referring providers, and patient characteristics. We analyzed themes from qualitative data to determine barriers and facilitators to HaH use. RESULTS: Our evaluation showed week-to-week increases in HaH capacity utilization during the multifaceted implementation strategy period, compared to discrete-period trends (slope-change odds ratio-1.02, 1.01-1.04). Counts of referring providers also increased week to week, compared to discrete-period trends (slope-change means ratio-1.05, 1.03-1.07). The increase in HaH utilization was largest among rural residents (11 to 34%). Barriers included HaH-related information gaps and referral challenges; facilitators included patient-centeredness of HaH care. CONCLUSIONS: A multifaceted implementation strategy was associated with increased HaH capacity utilization, provider adoption, and patient diversity. Health systems may consider similar, contextually relevant multicomponent approaches to equitably expand HaH.

Soft Skills: The Work of Communication and Persuasion Among Nurse Navigators in Hospital at Home Programs.

OBJECTIVE: To assess the role of soft skills in the work of Hospital at Home (HaH) nurse navigators. BACKGROUND: In HaH programs that employ them, nurse navigators are often responsible for identifying, assessing, referring, and educating potential HaH patients. The experiences of these navigators have gone understudied. METHODS: Researchers conducted semistructured interviews and observations with nurse navigators (n = 7) who collectively cover 14 North Carolina-based HaH sites. Navigators were asked to keep diaries of responses to directed questions. RESULTS: In their capacity as navigators, interviewees said they served several roles: intermediaries between hospital and HaH staff, interpreters of clinical knowledge for patients, and champions of, and educators for, the home-based program. The navigators noted that the interpersonal soft skills of building rapport, clear communication, and gentle persuasion were of the utmost importance in this work. CONCLUSIONS: The job descriptions of nurse navigators in HaH programs should fully reflect the breadth of their responsibilities, including time performing soft skilled labor. Also, training for these roles should include techniques to develop and refine these skills.

[18F]-(2S,4R)4-Fluoroglutamine PET Imaging of Glutamine Metabolism in Murine Models of Hepatocellular Carcinoma (HCC).

Purpose: Quantitative in vivo [18F]-(2S,4R)4-fluoroglutamine ([18F]4-FGln or more simply [18F]FGln) metabolic kinetic parameters are compared with activity levels of glutamine metabolism in different types of hepatocellular carcinoma (HCC). Methods: For this study, we used two transgenic mouse models of HCC induced by protooncogenes, MYC, and MET. Biochemical data have shown that tumors induced by MYC have increased levels of glutamine metabolism compared to those induced by MET. One-hour dynamic [18F]FGln PET data were acquired and reconstructed for fasted MYC mice (n = 11 tumors from 7 animals), fasted MET mice (n = 8 tumors from 6 animals), fasted FVBN controls (n = 8 normal liver regions from 6 animals), nonfasted MYC mice (n = 16 tumors from 6 animals), and nonfasted FVBN controls (n = 8 normal liver regions from 3 animals). The influx rate constants (K 1) using the one-tissue compartment model were derived for each tumor with the left ventricular blood pool input function. Results: Influx rate constants were significantly higher for MYC tumors (K 1 = 0.374 ± 0.133) than for MET tumors (K 1 = 0.141 ± 0.058) under fasting conditions (P = 0.0002). Rate constants were also significantly lower for MET tumors (K 1 = 0.141 ± 0.135) than normal livers (K 1 = 0.332 ± 0.179) under fasting conditions (P = 0.0123). Fasting conditions tested for MYC tumors and normal livers did not result in any significant difference with P values > 0.005. Conclusion: Higher influx rate constants corresponded to elevated levels of glutamine metabolism as determined by biochemical assays. The data showed that there is a distinctive difference in glutamine metabolism between MYC and MET tumors. Our study has demonstrated the potential of [18F]FGln PET imaging as a tool to assess glutamine metabolism in HCC tumors in vivo with a caution that it may not be able to clearly distinguish HCC tumors from normal liver tissue.

Effect of a Multicomponent Sepsis Transition and Recovery Program on Mortality and Readmissions After Sepsis: The Improving Morbidity During Post-Acute Care Transitions for Sepsis Randomized Clinical Trial.

OBJECTIVES: To evaluate whether a nurse navigator-led, multicomponent Sepsis Transition And Recovery program improves 30-day mortality and readmission outcomes after sepsis hospitalization. DESIG: n: Multisite pragmatic randomized clinical trial. SETTING: Three hospitals in North Carolina from January 2019 to March 2020. PATIENTS: Eligible patients hospitalized for suspected sepsis and deemed high-risk for mortality or readmission by validated internal risk models. INTERVENTIONS: Patients were randomized to receive usual care alone (i.e., routine transition support, outpatient care; n = 342) or additional Sepsis Transition And Recovery support (n = 349). The 30-day intervention involved a multicomponent transition service led by a nurse navigator through telephone and electronic health record communication to facilitate best practice postsepsis care strategies during and after hospitalization including: postdischarge medication review, evaluation for new impairments or symptoms, monitoring comorbidities, and palliative care approach when appropriate. Clinical oversight was provided by a Hospital Medicine Transition Services team. MEASUREMENTS AND MAIN RESULTS: The primary outcome was a composite of mortality or hospital readmission at 30 days. Logistic regression models were constructed to evaluate marginal and conditional odds ratios (adjusted for prognostic covariates: age, comorbidity, and organ dysfunction at enrollment). Among 691 randomized patients (mean age = 63.7 ± 15.1 yr; 52% female), a lower percentage of patients in the Sepsis Transition And Recovery group experienced the primary outcome compared with the usual care group (28.7% vs 33.3%; risk difference, 4.7%; odds ratio, 0.80; 95% CI, 0.58-1.11; adjusted odds ratio, 0.80; 95% CI, 0.64-0.98). There were 74 deaths (Sepsis Transition And Recovery: 33 [9.5%] vs usual care: 41 [12.0%]) and 155 rehospitalizations (Sepsis Transition And Recovery: 71 [20.3%] vs usual care: 84 [24.6%]). CONCLUSIONS: In a multisite randomized clinical trial of patients hospitalized with sepsis, patients provided with a 30-day program using a nurse navigator to provide best practices for postsepsis care experienced a lower proportion of either mortality or rehospitalization within 30 days after discharge. Further research is needed to understand the contextual factors associated with successful implementation.

Insights From Rapid Deployment of a "Virtual Hospital" as Standard Care During the COVID-19 Pandemic.

BACKGROUND: Pandemics disrupt traditional health care operations by overwhelming system resource capacity but also create opportunities for care innovation. OBJECTIVE: To describe the development and rapid deployment of a virtual hospital program, Atrium Health hospital at home (AH-HaH), within a large health care system. DESIGN: Prospective case series. SETTING: Atrium Health, a large integrated health care organization in the southeastern United States. PATIENTS: 1477 patients diagnosed with coronavirus disease 2019 (COVID-19) from 23 March to 7 May 2020 who received care via AH-HaH. INTERVENTION: A virtual hospital model providing proactive home monitoring and hospital-level care through a virtual observation unit (VOU) and a virtual acute care unit (VACU) in the home setting for eligible patients with COVID-19. MEASUREMENTS: Patient demographic characteristics, comorbid conditions, treatments administered (intravenous fluids, antibiotics, supplemental oxygen, and respiratory medications), transfer to inpatient care, and hospital outcomes (length of stay, intensive care unit [ICU] admission, mechanical ventilation, and death) were collected from electronic health record data. RESULTS: 1477 patients received care in either the AH-HaH VOU or VACU or both settings, with a median length of stay of 11 days. Of these, 1293 (88%) patients received care in the VOU only, with 40 (3%) requiring inpatient hospitalization. Of these 40 patients, 16 (40%) spent time in the ICU, 7 (18%) required ventilator support, and 2 (5%) died during their hospital admission. In total, 184 (12%) patients were ever admitted to the VACU, during which 21 patients (11%) required intravenous fluids, 16 (9%) received antibiotics, 40 (22%) required respiratory inhaler or nebulizer treatments, 41 (22%) used supplemental oxygen, and 24 (13%) were admitted as an inpatient to a conventional hospital. Of these 24 patients, 10 (42%) required ICU admission, 1 (3%) required a ventilator, and none died during their hospital admission. LIMITATION: Generalizability is limited to patients with a working telephone and the ability to comply with the monitoring protocols. CONCLUSION: Virtual hospital programs have the potential to provide health systems with additional inpatient capacity during the COVID-19 pandemic and beyond. PRIMARY FUNDING SOURCE: Atrium Health.

Protocol for a two-arm pragmatic stepped-wedge hybrid effectiveness-implementation trial evaluating Engagement and Collaborative Management to Proactively Advance Sepsis Survivorship (ENCOMPASS).

BACKGROUND: Sepsis survivors experience high morbidity and mortality, and healthcare systems lack effective strategies to address patient needs after hospital discharge. The Sepsis Transition and Recovery (STAR) program is a navigator-led, telehealth-based multicomponent strategy to provide proactive care coordination and monitoring of high-risk patients using evidence-driven, post-sepsis care tasks. The purpose of this study is to evaluate the effectiveness of STAR to improve outcomes for sepsis patients and to examine contextual factors that influence STAR implementation. METHODS: This study uses a hybrid type I effectiveness-implementation design to concurrently test clinical effectiveness and gather implementation data. The effectiveness evaluation is a two-arm, pragmatic, stepped-wedge cluster randomized controlled trial at eight hospitals in North Carolina comparing clinical outcomes between sepsis survivors who receive Usual Care versus care delivered through STAR. Each hospital begins in a Usual Care control phase and transitions to STAR in a randomly assigned sequence (one every 4 months). During months that a hospital is allocated to Usual Care, all eligible patients will receive usual care. Once a hospital transitions to STAR, all eligible patients will receive STAR during their hospitalization and extending through 90 days from discharge. STAR includes centrally located nurse navigators using telephonic counseling and electronic health record-based support to facilitate best-practice post-sepsis care strategies including post-discharge review of medications, evaluation for new impairments or symptoms, monitoring existing comorbidities, and palliative care referral when appropriate. Adults admitted with suspected sepsis, defined by clinical criteria for infection and organ failure, are included. Planned enrollment is 4032 patients during a 36-month period. The primary effectiveness outcome is the composite of all-cause hospital readmission or mortality within 90 days of discharge. A mixed-methods implementation evaluation will be conducted before, during, and after STAR implementation. DISCUSSION: This pragmatic evaluation will test the effectiveness of STAR to reduce combined hospital readmissions and mortality, while identifying key implementation factors. Results will provide practical information to advance understanding of how to integrate post-sepsis management across care settings and facilitate implementation, dissemination, and sustained utilization of best-practice post-sepsis management strategies in other heterogeneous healthcare delivery systems. TRIAL REGISTRATION: NCT04495946 . Submitted July 7, 2020; Posted August 3, 2020.

Predictive Accuracy of Quick Sequential Organ Failure Assessment for Hospital Mortality Decreases With Increasing Comorbidity Burden Among Patients Admitted for Suspected Infection.

OBJECTIVES: Evaluate the accuracy of the quick Sequential Organ Failure Assessment tool to predict mortality across increasing levels of comorbidity burden. DESIGN: Retrospective observational cohort study. SETTING: Twelve acute care hospitals in the Southeastern United States. PATIENTS: A total of 52,187 patients with suspected infection presenting to the Emergency Department between January 2014 and September 2017. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The primary outcome was hospital mortality. We used electronic health record data to calculate quick Sequential Organ Failure Assessment risk scores from vital signs and laboratory values documented during the first 24 hours. We calculated Charlson Comorbidity Index scores to quantify comorbidity burden. We constructed logistic regression models to evaluate differences in the performance of quick Sequential Organ Failure Assessment greater than or equal to 2 to predict hospital mortality in patients with no documented (Charlson Comorbidity Index = 0), low (Charlson Comorbidity Index = 1-2), moderate (Charlson Comorbidity Index = 3-4), or high (Charlson Comorbidity Index ≥ 5) comorbidity burden. Among the cohort, 2,030 patients died in the hospital (4%). No comorbidities were documented for 5,038 patients (10%), 9,235 patients (18%) had low comorbidity burden, 12,649 patients (24%) had moderate comorbidity burden, and 25,265 patients (48%) had high comorbidity burden. Overall model discrimination for quick Sequential Organ Failure Assessment greater than or equal to 2 was the area under the receiver operating characteristic curve of 0.71 (95% CI, 0.69-0.72). A model including both quick Sequential Organ Failure Assessment and Charlson Comorbidity Index had improved discrimination compared with Charlson Comorbidity Index alone (area under the receiver operating characteristic curve, 0.77; 95% CI, 0.76-0.78 vs area under the curve, 0.61; 95% CI, 0.59-0.62). Discrimination was highest among patients with no documented comorbidities (quick Sequential Organ Failure Assessment area under the receiver operating characteristic curve, 0.84; 95% CI; 0.79-0.89) and lowest among high comorbidity patients (quick Sequential Organ Failure Assessment area under the receiver operating characteristic curve, 0.67; 95% CI, 0.65-0.68). The strength of association between quick Sequential Organ Failure Assessment and mortality ranged from 30.5-fold increased likelihood in patients with no comorbidities to 4.7-fold increased likelihood in patients with high comorbidity. CONCLUSIONS: The accuracy of quick Sequential Organ Failure Assessment to predict hospital mortality diminishes with increasing comorbidity burden. Patients with comorbidities may have baseline abnormalities in quick Sequential Organ Failure Assessment variables that reduce predictive accuracy. Additional research is needed to better understand quick Sequential Organ Failure Assessment performance across different comorbid conditions with modification that incorporates the context of changes to baseline variables.

Aiming to Improve Readmissions Through InteGrated Hospital Transitions (AIRTIGHT): a Pragmatic Randomized Controlled Trial.

BACKGROUND: Despite years of intense focus, inpatient and observation readmission rates remain high and largely unchanged. Hospitals have little, robust evidence to guide the selection of interventions effective at reducing 30-day readmissions in real-world settings. OBJECTIVE: To evaluate if implementation of recent recommendations for hospital transition programs is effective at reducing 30-day readmissions in a population discharged to home and at high-risk for readmission. DESIGN: A non-blinded, pragmatic randomized controlled trial ( Clinicaltrials.gov : NCT02763202) conducted at two hospitals in Charlotte, North Carolina. PATIENTS: A total of 1876 adult patients, under the care of a hospitalist, and at high risk for readmissions. INTERVENTION: Random allocation to a Transition Services (TS) program (n = 935) that bridges inpatient, outpatient, and home settings, providing patients virtual and in-person access to a dedicated multidisciplinary team for 30-days, or usual care (n = 941). MAIN MEASURE: Thirty-day, unplanned, inpatient, or observation readmission rate. KEY RESULTS: The 30-day readmission rate was 15.2% in the TS group and 16.3% in the usual care group (RR 0.93; 95% [CI, 0.76 to 1.15]; P = 0.52). There were no significant differences in readmissions at 60 and 90 days or in 30-day Emergency Department visit rates. Patients, who were referred to TS and readmitted, had less Intensive Care Unit admissions 15.5% vs. 26.8% (RR 0.74; 95% [CI, 0.59 to 0.93]; P = 0.02). CONCLUSIONS: An intervention inclusive of contemporary recommendations does not reduce a high-risk population's 30-day readmission rate. The high crossover to usual care (74.8%) reflects the challenge of non-participation that is ubiquitous in the real-world implementation of population health interventions. TRIAL REGISTRY: ClinicalTrials.gov ; registration ID number: NCT02763202, URL: https://clinicaltrials.gov/ct2/show/NCT02763202.

Pharmacologic treatment of donor cells induced to have a Warburg effect-like metabolism does not alter embryonic development in vitro or survival during early gestation when used in somatic cell nuclear transfer in pigs.

Somatic cell nuclear transfer is a valuable technique for the generation of genetically engineered animals, however, the efficiency of cloning in mammalian species is low (1-3%). Differentiated somatic cells commonly used in nuclear transfer utilize the tricarboxylic acid cycle and cellular respiration for energy production. Comparatively the metabolism of somatic cells contrasts that of the cells within the early embryos which predominately use glycolysis. Early embryos (prior to implantation) are evidenced to exhibit characteristics of a Warburg Effect (WE)-like metabolism. We hypothesized that pharmacologically driven fibroblast cells can become more blastomere-like and result in improved in vitro embryonic development after SCNT. The goals were to determine if subsequent in vitro embryo development is impacted by (1) cloning pharmacologically treated donor cells pushed to have a WE-like metabolism or (2) culturing non-treated donor clones with pharmaceuticals used to push a WE-like metabolism. Additionally, we investigated early gestational survival of the donor-treated clone embryos. Here we demonstrate that in vitro development of clones is not hindered by pharmacologically treating either the donor cells or the embryos themselves with CPI, PS48, or the combination of these drugs. Furthermore, these experiments demonstrate that early embryos (or at least in vitro produced embryos) have a low proportion of mitochondria which have high membrane potential and treatment with these pharmaceuticals does not further alter the mitochondrial function in early embryos. Lastly, we show that survival in early gestation was not different between clones from pharmacologically induced WE-like donor cells and controls.

Longitudinal Evaluation of Myocardial Fatty Acid and Glucose Metabolism in Fasted and Nonfasted Spontaneously Hypertensive Rats Using MicroPET/CT.

Using longitudinal micro positron emission tomography (microPET)/computed tomography (CT) studies, we quantified changes in myocardial metabolism and perfusion in spontaneously hypertensive rats (SHRs), a model of left ventricular hypertrophy (LVH). Fatty acid and glucose metabolism were quantified in the hearts of SHRs and Wistar-Kyoto (WKY) normotensive rats using long-chain fatty acid analog 18F-fluoro-6-thia heptadecanoic acid (18F-FTHA) and glucose analog 18F-fluorodeoxyglucose (18F-FDG) under normal or fasting conditions. We also used 18F-fluorodihydrorotenol (18F-FDHROL) to investigate perfusion in their hearts without fasting. Rats were imaged at 4 or 5 times over their life cycle. Compartment modeling was used to estimate the rate constants for the radiotracers. Blood samples were obtained and analyzed for glucose and free fatty acid concentrations. SHRs demonstrated no significant difference in 18F-FDHROL wash-in rate constant ( P = .1) and distribution volume ( P = .1), significantly higher 18F-FDG myocardial influx rate constant ( P = 4×10-8), and significantly lower 18F-FTHA myocardial influx rate constant ( P = .007) than WKYs during the 2009-2010 study without fasting. SHRs demonstrated a significantly higher 18F-FDHROL wash-in rate constant ( P = 5×10-6) and distribution volume ( P = 3×10-8), significantly higher 18F-FDG myocardial influx rate constant ( P = 3×10-8), and a higher trend of 18F-FTHA myocardial influx rate constant (not significant, P = .1) than WKYs during the 2011-2012 study with fasting. Changes in glucose plasma concentrations were generally negatively correlated with corresponding radiotracer influx rate constant changes. The study indicates a switch from preferred fatty acid metabolism to increased glucose metabolism with hypertrophy. Increased perfusion during the 2011-2012 study may be indicative of increased aerobic metabolism in the SHR model of LVH.

Zygote injection of CRISPR/Cas9 RNA successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs.

The CRISPR/Cas9 genome editing tool has increased the efficiency of creating genetically modified pigs for use as biomedical or agricultural models. The objectives were to determine if DNA editing resulted in a delay in development to the blastocyst stage or in a skewing of the sex ratio. Six DNA templates (gBlocks) that were designed to express guide RNAs that target the transmembrane protease, serine S1, member 2 (TMPRSS2) gene were in vitro transcribed. Pairs of CRISPR guide RNAs that flanked the start codon and polyadenylated Cas9 were co-injected into the cytoplasm of zygotes and cultured in vitro to the blastocyst stage. Blastocysts were collected as they formed on days 5, 6 or 7. PCR was performed to determine genotype and sex of each embryo. Separately, embryos were surgically transferred into recipient gilts on day 4 of estrus. The rate of blastocyst development was not significantly different between CRISPR injection embryos or the non-injected controls at day 5, 6 or 7 (p = 0.36, 0.09, 0.63, respectively). Injection of three CRISPR sets of guides resulted in a detectable INDEL in 92-100 % of the embryos analyzed. There was not a difference in the number of edits or sex ratio of male to female embryos when compared between days 5, 6 and 7 to the controls (p > 0.22, >0.85). There were 12 resulting piglets and all 12 had biallelic edits of TMRPSS2. Zygote injection with CRISPR/Cas9 continues to be a highly efficient tool to genetically modify pig embryos.

Ictal lack of binding to brain parenchyma suggests integrity of the blood-brain barrier for 11C-dihydroergotamine during glyceryl trinitrate-induced migraine.

SEE DREIER DOI 101093/AWW112 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: For many decades a breakdown of the blood-brain barrier has been postulated to occur in migraine. Hypothetically this would facilitate access of medications, such as dihydroergotamine or triptans, to the brain despite physical properties otherwise restricting their entry. We studied the permeability of the blood-brain barrier in six migraineurs and six control subjects at rest and during acute glyceryl trinitrate-induced migraine attacks using positron emission tomography with the novel radioligand (11)C-dihydroergotamine, which is chemically identical to pharmacologically active dihydroergotamine. The influx rate constant Ki, average dynamic image and time activity curve were assessed using arterial blood sampling and served as measures for receptor binding and thus blood-brain barrier penetration. At rest, there was binding of (11)C-dihydroergotamine in the choroid plexus, pituitary gland, and venous sinuses as expected from the pharmacology of dihydroergotamine. However, there was no binding to the brain parenchyma, including the hippocampus, the area with the highest density of the highest-affinity dihydroergotamine receptors, and the raphe nuclei, a postulated brainstem site of action during migraine, suggesting that dihydroergotamine is not able to cross the blood-brain barrier. This binding pattern was identical in migraineurs during glyceryl trinitrate-induced migraine attacks as well as in matched control subjects. We conclude that (11)C-dihydroergotamine is unable to cross the blood-brain barrier interictally or ictally demonstrating that the blood-brain barrier remains tight for dihydroergotamine during acute glyceryl trinitrate-induced migraine attacks.

Evaluation of Molecular Methods for Identification of Salmonella Serovars.

Classification by serotyping is the essential first step in the characterization of Salmonella isolates and is important for surveillance, source tracking, and outbreak detection. To improve detection and reduce the burden of salmonellosis, several rapid and high-throughput molecular Salmonella serotyping methods have been developed.The aim of this study was to compare three commercial kits, Salm SeroGen (Salm Sero-Genotyping AS-1 kit), Check&Trace (Check-Points), and xMAP (xMAP Salmonella serotyping assay), to the Salmonella genoserotyping array (SGSA) developed by our laboratory. They were assessed using a panel of 321 isolates that represent commonly reported serovars from human and nonhuman sources globally. The four methods correctly identified 73.8% to 94.7% of the isolates tested. The methods correctly identified 85% and 98% of the clinically important Salmonella serovars Enteritidis and Typhimurium, respectively. The methods correctly identified 75% to 100% of the nontyphoidal, broad host range Salmonella serovars, including Heidelberg, Hadar, Infantis, Kentucky, Montevideo, Newport, and Virchow. The sensitivity and specificity of Salmonella serovars Typhimurium and Enteritidis ranged from 85% to 100% and 99% to 100%, respectively.It is anticipated that whole-genome sequencing will replace serotyping in public health laboratories in the future. However, at present, it is approximately three times more expensive than molecular methods. Until consistent standards and methodologies are deployed for whole-genome sequencing, data analysis and interlaboratory comparability remain a challenge. The use of molecular serotyping will provide a valuable high-throughput alternative to traditional serotyping. This comprehensive analysis provides a detailed comparison of commercial kits available for the molecular serotyping of Salmonella.

Biodistribution of Antibody-MS2 Viral Capsid Conjugates in Breast Cancer Models.

A variety of nanoscale scaffolds, including virus-like particles (VLPs), are being developed for biomedical applications; however, little information is available about their in vivo behavior. Targeted nanoparticles are particularly valuable as diagnostic and therapeutic carriers because they can increase the signal-to-background ratio of imaging agents, improve the efficacy of drugs, and reduce adverse effects by concentrating the therapeutic molecule in the region of interest. The genome-free capsid of bacteriophage MS2 has several features that make it well-suited for use in delivery applications, such as facile production and modification, the ability to display multiple copies of targeting ligands, and the capacity to deliver large payloads. Anti-EGFR antibodies were conjugated to MS2 capsids to construct nanoparticles targeted toward receptors overexpressed on breast cancer cells. The MS2 agents showed good stability in physiological conditions up to 2 days and specific binding to the targeted receptors in in vitro experiments. Capsids radiolabeled with 64Cu isotopes were injected into mice possessing tumor xenografts, and both positron emission tomography-computed tomography (PET/CT) and scintillation counting of the organs ex vivo were used to determine the localization of the agents. The capsids exhibit surprisingly long circulation times (10-15% ID/g in blood at 24 h) and moderate tumor uptake (2-5% ID/g). However, the targeting antibodies did not lead to increased uptake in vivo despite in vitro enhancements, suggesting that extravasation is a limiting factor for delivery to tumors by these particles.

Chronic methamphetamine exposure prior to middle cerebral artery occlusion increases infarct volume and worsens cognitive injury in Male mice.

Emerging evidence indicates that methamphetamine (MA) abuse can impact cardiovascular disease. In humans, MA abuse is associated with an increased risk of stroke as well as an earlier age at which the stroke occurs. However, little is known about how chronic daily MA exposure can impact ischemic outcome in either humans or animal models. In the present study, mice were injected with MA (10 mg/kg, i.p.) or saline once daily for 10 consecutive days. Twenty-four hours after the final injection, mice were subjected to transient middle cerebral artery occlusion (tMCAO) for one hour followed by reperfusion. Mice were tested for novel object memory at 96 h post-reperfusion, just prior to removal of brains for quantification of infarct volume using 2,3,5-Triphenyltetrazolium Chloride (TTC) staining. Mice treated with MA prior to tMCAO showed decreased object memory recognition and increased infarct volume compared to saline-treated mice. These findings indicate that chronic MA exposure can worsen both cognitive and morphological outcomes following cerebral ischemia.

Imaging a functional tumorigenic biomarker in the transformed epithelium.

Proteases responsible for the increased peritumoral proteolysis associated with cancer represent functional biomarkers for monitoring tumorigenesis. One attractive extracellular biomarker is the transmembrane serine protease matriptase. Found on the surface of epithelial cells, the activity of matriptase is regulated by its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). Quantitative mass spectrometry allowed us to show that, in selected cancers, HAI-1 expression decreases, leading to active matriptase. A preclinical probe specific for the measurement of emergent active matriptase was developed. Using an active-site-specific, recombinant human antibody for matriptase, we found that the selective targeting of active matriptase can be used to visualize the tumorigenic epithelium. Live-cell fluorescence imaging validated the selectivity of the antibody in vitro by showing that the probe localized only to cancer cell lines with active matriptase on the surface. Immunofluorescence with the antibody documented significant levels of active matriptase in 68% of primary and metastatic colon cancer sections from tissue microarrays. Labeling of the active form of matriptase in vivo was measured in human colon cancer xenografts and in a patient-derived xenograft model using near-infrared and single-photon emission computed tomography imaging. Tumor uptake of the radiolabeled antibody, (111)In-A11, by active matriptase was high in xenografts (28% injected dose per gram) and was blocked in vivo by the addition of a matriptase-specific variant of ecotin. These findings suggest, through a HAI-1-dependent mechanism, that emergent active matriptase is a functional biomarker of the transformed epithelium and that its proteolytic activity can be exploited to noninvasively evaluate tumorigenesis in vivo.

The cytosolic adaptor AP-1A is essential for the trafficking and function of Niemann-Pick type C proteins.

Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by over-accumulation of low-density lipoprotein-derived cholesterol and glycosphingolipids in late endosomes/lysosomes (LE/L) throughout the body. Human mutations in either NPC1 or NPC2 genes have been directly associated with impaired cholesterol efflux from LE/L. Independent from its role in cholesterol homeostasis and its NPC2 partner, NPC1 was unexpectedly identified as a critical player controlling intracellular entry of filoviruses such as Ebola. In this study, a yeast three-hybrid system revealed that the NPC1 cytoplasmic tail directly interacts with the clathrin adaptor protein AP-1 via its acidic/di-leucine motif. Consequently, a nonfunctional AP-1A cytosolic complex resulted in a typical NPC-like phenotype mainly due to a direct impairment of NPC1 trafficking to LE/L and a partial secretion of NPC2. Furthermore, the mislocalization of NPC1 was not due to cholesterol accumulation in LE/L, as it was not rescued upon treatment with Mß-cyclodextrin, which almost completely eliminated intracellular free cholesterol. Our cumulative data demonstrate that the cytosolic clathrin adaptor AP-1A is essential for the lysosomal targeting and function of NPC1 and NPC2.

Regulatory CD8(+)CD122 (+) T-cells predominate in CNS after treatment of experimental stroke in male mice with IL-10-secreting B-cells.

Clinical stroke induces inflammatory processes leading to cerebral and splenic injury and profound peripheral immunosuppression. IL-10 expression is elevated during major CNS diseases and limits inflammation in the brain. Recent evidence demonstrated that transfer of IL-10(+) B-cells reduced infarct volume in male C57BL/6J (wild-type, WT) recipient mice when given 24 h prior to or 4 h after middle cerebral artery occlusion (MCAO). The purpose of this study was to determine if passively transferred IL-10(+) B-cells can exert therapeutic and immunoregulatory effects when injected 24 h after MCAO induction in B-cell-sufficient male WT mice. The results demonstrated that IL-10(+) B-cell treated mice had significantly reduced infarct volumes in the ipsilateral cortex and hemisphere and improved neurological deficits vs. Vehicle-treated control mice after 60 min occlusion and 96 h of reperfusion. The MCAO-protected B-cell recipient mice had less splenic atrophy and reduced numbers of activated, inflammatory T-cells, decreased infiltration of T-cells and a less inflammatory milieu in the ischemic hemispheres compared with Vehicle-treated control mice. These immunoregulatory changes occurred in concert with the predominant appearance of IL-10-secreting CD8(+)CD122(+) Treg cells in both the spleen and the MCAO-affected brain hemisphere. This study for the first time demonstrates a major neuroprotective role for IL-10(+) B-cells in treating MCAO in male WT mice at a time point well beyond the ~4 h tPA treatment window, leading to the generation of a dominant IL-10(+)CD8(+)CD122(+) Treg population associated with spleen preservation and reduced CNS inflammation.