Effects of prostaglandins E2 and F2α on electromyogram of cat colonin vitro

Prostaglandins cause diarrhea, and their production by the gut increases in diarrheal states. We studied the effects of PGF₂ and PGE₂ on the electromyogram recorded from the cat colonin vitro to determine if these prostaglandins might produce electromyographic changes similar to those seen in diarrheal states. PGF₂ decreased slow wave frequency and uncoupled slow wave propagation in the proximal colon. It increased the frequency of migrating spike bursts. PGE₂ had no effect on slow waves, but increased the frequency of the migrating spike burst. PGF₂ produced electromyographic changes similar to those recorded from the colon of cats with spontaneous diarrhea or after exposure to diarrhea-producing agents such as ricinoleate or quinidine. Some diarrhea-producing agents are likely to act by increasing prostaglandin production.     

Altered gastric emptying and prevention of radiation-induced vomiting in dogs

The relation between radiation-induced vomiting and gastric emptying is unclear and the treatment of this condition is not established. We explored, therefore, (a) the effect of cobalt 60 irradiation on gastric emptying of solids and liquids and (b) the possibility of preventing radiation-induced vomiting with the dopamine antagonist, domperidone. Twenty dogs were studied on two separate days, blindly and in random order, after i.v. injection of either a placebo or 0.06 mg/kg domperidone. On a third day, they received 8 Gy (800 rads) whole body irradiation with cobalt 60 gamma-rays after either placebo (n = 10) or domperidone (n = 10). Before each study, each dog was fed chicken liver tagged in vivo with 99mTc-sulfur colloid (solid marker), and water containing 111In-diethylenetriamine pentaacetic acid (liquid marker). Dogs were placed in a Pavlov stand for the subsequent 3 h and radionuclide imaging was performed at 10-min intervals. Irradiation produced vomiting in 9 of 10 dogs given placebo but only in 1 of 10 dogs pretreated with domperidone (p less than 0.01). Gastric emptying of liquids and solids was significantly suppressed by irradiation (p less than 0.01) after both placebo and domperidone. These results demonstrate that radiation-induced vomiting is accompanied by suppression of gastric emptying. Furthermore, domperidone prevents vomiting produced by ionizing radiation but does not alter the accompanying delay of gastric emptying.     

Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis

Vitamin C (L-ascorbic acid; AsA) acts as a potent antioxidant and cellular reductant in plants and animals. AsA has long been known to have many critical physiological roles in plants, yet its biosynthesis is only currently being defined. A pathway for AsA biosynthesis that features GDP-mannose and L-galactose has recently been proposed for plants. We have isolated a collection of AsA-deficient mutants of Arabidopsis thaliana that are valuable tools for testing of an AsA biosynthetic pathway. The best-characterized of these mutants (vtc1) contains approximately 25% of wild-type AsA and is defective in AsA biosynthesis. By using a combination of biochemical, molecular, and genetic techniques, we have demonstrated that the VTC1 locus encodes a GDP-mannose pyrophosphorylase (mannose-1-P guanyltransferase). This enzyme provides GDP-mannose, which is used for cell wall carbohydrate biosynthesis and protein glycosylation as well as for AsA biosynthesis. In addition to genetically defining the first locus involved in AsA biosynthesis, this work highlights the power of using traditional mutagenesis techniques coupled with the Arabidopsis Genome Initiative to rapidly clone physiologically important genes.     

Effects of lethal irradiation in zebrafish and rescue by hematopoietic cell transplantation

The study of hematopoiesis has been greatly facilitated by transplantation of blood cell populations into recipient animals. Efficient engraftment of donor cells generally requires ablation of the host hematopoietic system. The zebrafish has recently emerged as a developmental and genetic system to study hematopoiesis. To enable the study of hematopoietic stem cell (HSC) biology, immune cell function, and leukemogenesis in zebrafish, we have developed hematopoietic cell transplantation (HCT) into adult recipient animals conditioned by gamma irradiation. Dose-response experiments showed that the minimum lethal dose (MLD) of 40 Gy led to the specific ablation of hematolymphoid cells and death by 14 days after irradiation. Sublethal irradiation doses of 20 Gy predominantly ablated lymphocytes and permitted transplantation of a lethal T-cell leukemia. Finally, transplantation of hematopoietic cells carrying transgenes yielding red fluorescent erythrocytes and green fluorescent leukocytes showed that HCT is sufficient to rescue the MLD, that recipient hematolymphoid tissues were repopulated by donor-derived cells, and that donor blood cell lineages can be independently visualized in living recipients. Together, these results establish transplantation assays to test for HSC function and oncogenic transformation in zebrafish.     

Complete cervical spinal cord injury above C6 predicts the need for tracheostomy

Background

Failed extubation and delayed tracheostomy contribute to poor outcomes in patients with a traumatic spinal cord injury (SCI). We determined if the level and completeness of SCI predict the need for tracheostomy.

Methods

Data from 256 patients with SCI between C1 and T3 with or without tracheostomy were retrospectively analyzed. Logistic regression identified predictors for tracheostomy. Data are presented as raw percentage or odds ratio (OR) with 95% confidence interval. P < .05 indicates significance.

Results

Complete spinal cord injuries were common in patients requiring tracheostomy (55% vs 18%, P < .05), and predicted the need for tracheostomy (OR: 6.4 (3.1 to 13.5), P < .05). An injury above C6 predicted the need for tracheostomy in patients with complete injury (OR: 3.7 (1 to 11.9), P < .05), but not incomplete injury (OR: .7 (.3 to 1.9); P = .53).

Conclusion

Tracheostomy is unlikely in patients with incomplete SCI, regardless of the level of injury. Patients with complete SCI above C6 are likely to require tracheostomy.     

Implementing deprescribing guidelines into frontline practice: Barriers and facilitators

A Bruyère Evidence-Based Deprescribing Guideline Symposium was held in March 2018; one component focused on implementing deprescribing guidelines into practice. An interactive discussion activity allowed the 107 participants to share experiences and ideas concerning the barriers and facilitators that arise when moving deprescribing guidelines into frontline practice. Participants identified 8 broad challenges and problem areas. These included challenges and barriers that arise in the daily practices of pharmacists and prescribers and in other health care settings, and those related to existing policies, processes, and financial structures. They also identified 10 factors that facilitated implementation efforts, including: educating patients, caregivers, health care providers (HCPs) and staff; improving collaboration across practice disciplines; expanding the evidence for deprescribing; and fostering organizational cultures of deprescribing. The results indicate that participants are committed to deprescribing and are moving forward with efforts to bring about change. Participants recognize that the implementation of deprescribing is best conceived of as a comprehensive systems change, and that patients and the public need to be involved in deprescribing processes and activities.     

Deprescribing: Future directions for research

A World Café workshop was held at the Bruyère Evidence-Based Deprescribing Guidelines Symposium in March 2018 with 30 participants (researchers, clinicians, policy makers, stakeholders). This workshop explored priorities for future work in the field of deprescribing and deprescribing guidelines through group discussion. The discussions were guided by the following questions: (1) What are deprescribing research priorities (to inform guideline development), (2) What outcome measures are important for developing deprescribing guidelines, and (3) How do we evaluate the implementation and effectiveness of deprescribing guidelines? Discussion from all 3 questions identified 6 main priority areas: (1) conducting high-quality and long-term clinical trials that measure patient-important outcomes, (2) focusing on patient involvement and perspectives, (3) investigating the pharmacoeconomics of deprescribing interventions, (4) understanding deprescribing interventions in different populations, (5) generating evidence on clinical management during deprescribing (e.g. managing adverse drug withdrawal effects, subsequent re-prescribing), and (6) implementing interventions in clinical practice. These topics represent what a group of experienced researchers, clinicians, and stakeholders in the field collectively felt was important to consider for design and implementation of future deprescribing studies. The aim is for these findings to stimulate future discussions and be considered by granting agencies, policy makers, deprescribing research networks, and individual researchers planning future deprescribing studies.     

Synthetic control of mammalian-cell motility by engineering chemotaxis to an orthogonal bioinert chemical signal

Directed migration of diverse cell types plays a critical role in biological processes ranging from development and morphogenesis to immune response, wound healing, and regeneration. However, techniques to direct, manipulate, and study cell migration in vitro and in vivo in a specific and facile manner are currently limited. We conceived of a strategy to achieve direct control over cell migration to arbitrary user-defined locations, independent of native chemotaxis receptors. Here, we show that genetic modification of cells with an engineered G protein-coupled receptor allows us to redirect their migration to a bioinert drug-like small molecule, clozapine-N-oxide (CNO). The engineered receptor and small-molecule ligand form an orthogonal pair: The receptor does not respond to native ligands, and the inert drug does not bind to native cells. CNO-responsive migration can be engineered into a variety of cell types, including neutrophils, T lymphocytes, keratinocytes, and endothelial cells. The engineered cells migrate up a gradient of the drug CNO and transmigrate through endothelial monolayers. Finally, we demonstrate that T lymphocytes modified with the engineered receptor can specifically migrate in vivo to CNO-releasing beads implanted in a live mouse. This technology provides a generalizable genetic tool to systematically perturb and control cell migration both in vitro and in vivo. In the future, this type of migration control could be a valuable module for engineering therapeutic cellular devices.The ability of many cell types to migrate long distances within the body and specifically localize to target sites of action is critical for their proper function. For example, immune cells rapidly home to sites of infection, concentrating their powerful cytotoxic and proinflammatory activities for maximum efficacy while limiting damage to healthy tissue. In morphogenesis, cells undergo a complex stereotyped process involving migration as well as proliferation, differentiation, and programmed cell death to produce fully developed multicellular structures. In wound healing and regenerative processes, stem and progenitor cells home to injured tissues from nearby sites—as well as from distant locations including the bone marrow—to provide a stream of new cells to replenish and provide trophic support to old and damaged cells.Cell migration is also an important factor to consider in the use of cells as therapeutic agents. The use of cells for the treatment of a growing array of diseases including cancer, autoimmunity, and chronic wounds is currently being explored (1–6). The appropriate and efficient localization of therapeutic cells to sites of disease has been identified as an important factor for successful cell-based therapy (7–17). However, preclinical studies and clinical trials to date have shown that the homing to sites of disease of many cell types commonly used as therapeutics is frequently impaired or limited, especially after ex vivo expansion of cells in culture (7, 12, 18, 19).The ability to redirect the migration of cells to any user-specified location in the body would be a powerful enabling technology for basic research as well as for future applications, but there are currently few easily generalizable strategies to accomplish this goal. We conceived of an approach to direct cellular homing to small molecules by expressing, in motile cells, engineered G protein-coupled receptors (GPCRs) called receptors activated solely by a synthetic ligand (RASSLs) (20, 21).RASSLs are engineered to be unresponsive to endogenous ligands but can be activated by pharmacologically inert orthogonal small molecules (Fig. 1A). Versions of these receptors exist for the three major GPCR signaling pathways (Gαs-, Gαi-, and Gαq-coupled receptors), and the design of a new arrestin-biased variant has recently been reported (21, 22). Because GPCRs control many important physiological functions, including cell migration, we hypothesized that, by expressing these engineered receptors in motile cells, we could develop a general strategy for establishing user control over cell homing (Fig. 1B). Here, we use a family of second-generation RASSLs, known as designer receptors exclusively activated by a designer drug (DREADDs), that are activated only by the small molecule clozapine-N-oxide (CNO), an inert metabolite of the FDA-approved antipsychotic drug clozapine (Fig. S1) (20). CNO is highly bioavailable in rodents and humans, lacks affinity for any known receptors, channels, and transporters, and does not cause any appreciable physiological effects when systemically administered in normal mice (20, 23, 24).Open in a separate windowFig. 1.Engineered Gαi-coupled GPCRs Di3 and Di mediate cytoskeletal changes and chemotaxis of HL-60 neutrophils in response to CNO. (A) RASSLs are engineered GPCRs that interact orthogonally with a bioinert small-molecule drug. Natural ligands do not interact with the engineered receptors, and the bioinert drug that activates the engineered receptors does not interact with native receptors. (B) We tested whether certain second-generation RASSLs known as DREADDs could mediate cell motility. (C) Changes in electrical impedance that result from cell spreading in response to drug or ligand are detected by an electrode array. HL-60 neutrophils transiently transfected to express engineered GPCRs were plated on fibronectin-coated impedance assay plates and stimulated with vehicle control, 100 nM fMLP (positive control chemoattractant) or 100 nM CNO. All cells responded to fMLP whereas only Di3- or Di-expressing cells responded to CNO. Mean ± SEM for n = 3 replicates is shown. (D) Cell migration of HL-60 neutrophils transiently transfected with engineered GPCRs was quantitated in a porous transwell Boyden-chamber assay. All cells migrated in response to fMLP whereas only Di3- or Di-expressing cells migrated in response to CNO. Drug concentrations used: 100 nM CNO, 100 nM fMLP. Mean ± SEM for n = 3 replicates is shown. (E) Polarization and cell migration in neutrophils involves Rac and PI3K activation. Di-expressing HL-60 neutrophils were treated with 100 nM fMLP or 100 nM CNO before immunoblotting for phosphorylated Akt and phosphorylated PAK as readouts for PI3K and Rac activity, respectively. Peak levels of phospho-Akt and phospho-PAK are shown for each condition. Both were increased by CNO stimulation in Di cells but not in control cells (P < 0.01 by Student t test). Stimulation with fMLP increased phospho-Akt and phospho-PAK levels in both Di and control cells (P < 0.01 by Student t test), but Di cells showed higher peak levels of phospho-Akt than did control cells (P < 0.01 by Student t test). Three (for CNO) or four (for fMLP) independent experiments were performed and mean ± SEM are shown.     

Trends in Childhood Influenza Vaccination Coverage—U.S., 2004–2012

Objective

We compared estimates of childhood influenza vaccination coverage by health status, age, and racial/ethnic group across eight consecutive influenza seasons (2004 through 2012) based on two survey systems to assess trends in childhood influenza vaccination coverage in the U.S.

Methods

We used National Health Interview Survey (NHIS) and National Immunization Survey-Flu (NIS-Flu) data to estimate receipt of at least one dose of influenza vaccination among children aged 6 months to 17 years based on parental report. We computed estimates using Kaplan-Meier survival analysis methods.

Results

Based on the NHIS, overall influenza vaccination coverage with at least one dose of influenza vaccine among children increased from 16.2% during the 2004–2005 influenza season to 47.1% during the 2011–2012 influenza season. Children with health conditions that put them at high risk for complications from influenza had higher influenza vaccination coverage than children without these health conditions for all the seasons studied. In seven of the eight seasons studied, there were no significant differences in influenza vaccination coverage between non-Hispanic black and non-Hispanic white children. Influenza vaccination coverage estimates for children were slightly higher based on NIS-Flu data compared with NHIS data for the 2010–2011 and 2011–2012 influenza seasons (4.1 and 4.4 percentage points higher, respectively); both NIS-Flu and NHIS estimates had similar patterns of decreasing vaccination coverage with increasing age.

Conclusions

Although influenza vaccination coverage among children continued to increase, by the 2011–2012 influenza season, only slightly less than half of U.S. children were vaccinated against influenza. Much improvement is needed to ensure all children aged ≥6 months are vaccinated annually against influenza.Recommendations to vaccinate children against influenza began in 1960, when people with certain health conditions that put them at increased risk of severe illness from influenza were recommended to receive annual influenza vaccination, implicitly including children with high-risk conditions.¹ The recommendations for influenza vaccination of children remained unchanged until 2002, when providers were encouraged to vaccinate all children aged 6–23 months, regardless of medical conditions,² and in 2004, when all children aged 6–23 months were explicitly recommended for vaccination.³ In 2006, the influenza recommendations were expanded to include annual vaccination for all children aged 6–59 months.⁴ Recommendations were further expanded in 2008 to include annual vaccination of all children aged 6 months to 18 years.⁵Since the 2010–2011 influenza season, annual influenza vaccination has been recommended for all people aged 6 months and older.⁶ Children aged 6 months to 8 years should receive two doses of influenza vaccine, spaced four weeks apart, during their first season of vaccination and then one dose per season in subsequent seasons.⁷ During inter-pandemic seasons through 2011–2012, trivalent influenza vaccine was available, providing protection against two influenza A subtypes and one type B strain. During the 2009–2010 influenza season, two influenza vaccines were recommended: the trivalent seasonal influenza vaccination and the pandemic influenza A(H1N1)pdm09 (pH1N1) monovalent vaccination.⁸The National Health Interview Survey (NHIS) and the National Immunization Survey-Flu (NIS-Flu) have been the primary surveys used to measure influenza vaccination coverage among children. NHIS, an in-person household survey, began collecting parental report of influenza vaccination in 2005.⁹ NHIS has been considered the most representative source for estimates of influenza vaccination coverage among children aged 6 months to 17 years and has served as the Healthy People data source for influenza vaccination estimates.¹⁰ NHIS, however, has not routinely allowed for state-level estimates and is not timely enough to enable the reporting of influenza estimates before the beginning of the next influenza season. NIS-Flu is an ongoing telephone survey of households with children aged 6 months to 17 years. National and state-level estimates of influenza vaccination coverage for children based on parental-reported vaccination status from NIS-Flu have been reported by fall of the subsequent influenza vaccination season.^11,12The aims of this study were to (1) examine estimates of childhood influenza vaccination coverage over time by age, race/ethnicity, and high-risk status; and (2) compare estimates from NHIS and NIS-Flu, the two main survey systems currently used to measure influenza vaccination coverage among children in the U.S.