Recombinant adeno-associated virus-mediated global anterograde delivery of glial cell line-derived neurotrophic factor to the spinal cord: comparison of rubrospinal and corticospinal tracts in the rat

Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of spinal lower motoneurons. Gene delivery is a promising strategy to deliver therapeutic molecules to these vulnerable cells. However, definition of an optimal route of delivery capable of accessing neurons over a considerable extent of the neuraxis represents a significant logistical problem. Intramuscular vector injections are not ideal as this approach would involve hundreds of injections to completely treat an ALS patient and also would be dependent on retrograde transport of the viral platform of choice. Alternatively, upper motoneurons could deliver trophic factors over considerable distances by anterograde transport after a relatively localized intracerebral injection. To test this approach, the present study was designed to compare the corticospinal (CST) and rubrospinal (RST) tracts for their ability to transport recombinant adeno-associated virus serotype 5 (rAAV5)-derived green fluorescent protein (GFP) or glial cell line-derived neurotrophic factor (GDNF) to the spinal cord. Unilateral injections of rAAV5-GFP into the red nucleus (RN) or motor cortex of normal rats produced GFP-positive fibers in the appropriate descending tracts extending to the lumbar spinal cord. For both tracts, GFP-positive axonal projections into the spinal gray matter were consistently observed. GDNF immunohistochemistry demonstrated that confirmed RN injections resulted in GDNF-positive fibers projecting into spinal gray matter as seen in the GFP group. In contrast, confirmed cortical rAAV5-GDNF injections resulted in less evident staining in spinal cord. Spinal cord GDNF levels were elevated at distances up to 72 mm from the injection sites, and confirmed that RST-related GDNF transport to spinal cord surpassed CST-associated delivery.     

Kindness kills: the negative impact of pain as the fifth vital sign

BACKGROUND: The current emphasis on pain assessment as the fifth vital sign and the use of unscientific pain scales is causing serious injury and death from overmedication. STUDY DESIGN: This premise was tested by reviewing the case reports of all trauma center site surveys performed by the authors for the American College of Surgeons Committee on Trauma verification program during 2 separate time periods: 1994 through 1998 and 2000 through 2004. A total of 2,907 and 2,282 reports summarized by one of the authors, plus a total of 53 and 50 other reviewers, respectively, were analyzed from the records of 120 and 94 trauma centers. Most patients were men (71% and 66%) and had sustained blunt injury (83% and 79%). Average age was 35 years for both periods, with a range of 3 weeks to 97 years and 3 days to 98 years, respectively. The most common injuries involved head (33% and 34%), chest (13% and 13%), abdominal (22% and 21%), orthopaedic (18% and 18%), or multiple (9% and 14%). There were 1,459 and 867 deaths, respectively; all had a multidisciplinary peer review. RESULTS: Overmedication with sedatives/narcotics, during the two periods, clearly contributed to deaths in 13 and 32 patients and probably contributed to deaths in 5 and 14 patients, respectively. This occurred in 17 and 43 patients, respectively, after blunt injury and in 1 and 3 patients, respectively, after penetrating injury. Two clinical scenarios predominated, ie, overmedication in preparation for an imaging study and overmedication after discharge from ICU to the floor. The sequel of hypotension and compromised airway requiring intubation initiated a cascade of negative events that led to death. One patient in each period died as a result of prehospital overmedication. CONCLUSIONS: The current assessment of pain by computer-stored pain scales is in a state of imbalance, with excessive emphasis on undermedication at the same time ignoring overmedication. This imbalance reflects pain-service attempts to comply with external accrediting agencies. This preventable cause of death and disability in trauma patients is also occurring in noninjured patients. Surgeons must correct this problem by insisting on a balanced assessment of overmedication versus undermedication.     

Opportunities for improving post-hospital home medication management among older adults

Effective post-hospital home medication management among older adults is a convoluted, error-prone process. Older adults, whose complex medication regimens are often changed at hospital discharge, are susceptible to medication-related problems (e.g. Adverse Drug Events or ADEs) as they resume responsibility for managing their medications at home. Human error theory frames the discussion of multi-faceted, interacting factors including care system functions, like discharge medication teaching that contribute to post-hospital ADEs. The taxonomy and causes of post-hospital ADEs and related risk factors are reviewed, as we describe in high-risk older adults a population that may benefit from targeted interventions. Potential solutions and future research possibilities highlight the importance of interdisciplinary teams, involvement of clinical pharmacists, use of transitional care models, and improved use of informational technologies.     

Early detection of cystic fibrosis lung disease: multiple-breath washout versus raised volume tests

BACKGROUND: Lung clearance index (LCI), a measure of ventilation inhomogeneity derived from the multiple-breath inert gas washout (MBW) technique, has been shown to detect abnormal lung function more readily than spirometry in preschool children with cystic fibrosis, but whether this holds true during infancy is unknown. OBJECTIVES: To compare the extent to which parameters derived from the MBW and the raised lung volume rapid thoraco-abdominal compression (RVRTC) techniques identify diminished airway function in infants with cystic fibrosis when compared with healthy controls. METHODS: Measurements were performed during quiet sleep, with the tidal breathing MBW technique being performed before the forced expiratory manoeuvres. RESULTS: Measurements were obtained in 39 infants with cystic fibrosis (mean (SD) age 41.4 (22.0) weeks) and 21 controls (37.0 (15.1) weeks). Infants with cystic fibrosis had a significantly higher respiratory rate (38 (10) vs 32 (5) bpm) and LCI (8.4 (1.5) vs 7.2 (0.3)), and significantly lower values for all forced expiratory flow-volume parameters compared with controls. Girls with cystic fibrosis had significantly lower forced expiratory volume (FEV(0.5) and FEF(25-75 )) than boys (mean (95% CI girls-boys): -1.2 (-2.1 to -0.3) for FEV(0.5) Z score; FEF(25-75): -1.2 (-2.2 to -0.15)). When using both the MBW and RVRTC techniques, abnormalities were detected in 72% of the infants with cystic fibrosis, with abnormalities detected in 41% using both techniques and a further 15% by each of the two tests performed. CONCLUSIONS: These findings support the view that inflammatory and/or structural changes in the airways of children with cystic fibrosis start early in life, and have important implications regarding early detection and interventions. Monitoring of early lung disease and functional status in infants and young children with cystic fibrosis may be enhanced by using both MBW and the RVRTC.     

Characteristics of school campuses and physical activity among youth

BACKGROUND: Previous research suggests that school characteristics may influence physical activity. However, few studies have examined associations between school building and campus characteristics and objective measures of physical activity among middle school students. METHODS: Students from ten middle schools (n=248, 42% female, mean age 13.7 years) wore TriTrac-R3D accelerometers in 1997 recording measures of minute-by-minute physical movements during the school day that were then averaged over 15-minute intervals (n=16,619) and log-transformed. School characteristics, including school campus area, play area, and building area (per student) were assessed retrospectively in 2004-2005 using land-use parcel data, site visits, ortho-photos, architectural plans, and site maps. In 2006, linear mixed models using SAS PROC MIXED were fit to examine associations between school environmental variables and physical activity, controlling for potentially confounding variables. RESULTS: Area per enrolled student ranged from 8.8 to 143.7 m2 for school campuses, from 12.1 to 24.7 m2 for buildings, and from 0.4 to 58.9 m2 for play areas. Play area comprised from 3% to 62% of total campus area across schools. In separate regression models, school campus area per student (beta=0.2244, p<0.0001); building area per student (beta=2.1302, p<0.02); and play area per student (beta=0.347, p<0.0001) were each directly associated with log-TriTrac-R3D vector magnitude. Given the range of area density measures in this sample of schools, this translates into an approximate 20% to 30% increase in average vector magnitude, or walking 2 extra miles over the course of a week. CONCLUSIONS: Larger school campuses, school buildings, and play areas (per enrolled student) are associated with higher levels of physical activity in middle school students.     

Immunolocalization of the multi-sarco/endoplasmic reticulum Ca2+ATPase system in human platelets

We recently identified a multi-SERCA (sarco/endoplasmic reticulum Ca²⁺ ATPase) system in haemopoietic cells comprising the SERCA 2b, SERCA 3 and a new monoclonal anti-Ca²⁺ ATPase antibody (PL/IM 430) recognizable SERCA isoforms. We have now investigated the subcellular localization of these enzymes in human platelets by Western blotting of subcellular membrane fractions and by immunoelectron microscopy. We precisely defined the recognition specificity of the polyclonal anti-SERCA 2b, anti-SERCA 3, anti-SERCA 1 antibodies as well as of the monoclonal antibody PL/IM 430 by testing their recognition of the tryptic fragments of the SERCA isoforms. The analysis of fragmented membranes enriched in plasma membrane and intracellular membrane components by Western blotting showed that the SERCA 2b and the SERCA 3 isoforms were found in both the plasma membrane and the intracellular membrane fractions, whereas the PL/IM 430 recognizable SERCA isoform was restricted to membranes associated with the plasma membrane fraction. The immunoelectron microscopical study of the SERCA isoforms in resting platelets showed that: (i) the SERCA 2b isoform was expressed in membranes associated with the plasma membrane and open canalicular system, some α-granules and in unidentified membranes; (ii) the SERCA 3 isoform was found associated with plasma and intracellular membranes; and (iii) the PL/IM 430 recognizable SERCA isoform was observed only in structures associated with the cytoplasmic face of the plasma membranes, as confirmed by flow cytometry. Finally, since the PL/IM 430 antibody was raised against intracellular membranes, we looked for a potential membrane redistribution during the isolation procedure used for the preparation of the immunizing membranes. Neuraminidase treatment indeed induced a translocation of the PL/IM 430 recognizable SERCA isoform from plasma to intracellular membranes. Thus, the multi-SERCA system in platelets: (i) is distributed over different platelet membranes, (ii) presents a sub-compartmental organization with some overlapping, and (iii) is partly associated with motile membranes, reflecting an unrecognized level of complexity of Ca²⁺ stores in these cells.