A reunification of the US ("NIH") and International Unit into a single standard for Thrombin

The existence of two different units for Thrombin in widespread international use has caused confusion for many years. The holders of the WHO International Standard (IS) for Alpha Thrombin and the US Standard (also known as the "NIH Standard") now report on a collaboration to reunite the International Unit (IU) and the US unit ("NIH unit"). A study was organised involving 25 laboratories in 15 countries to investigate the possibility of preparing a common Standard with a common unit and to investigate aspects of methodology that cause divergence of results using the IS and US Standard. Laboratories were asked to measure the potency of two candidate replacement standards (C, 01/578 and D, 01/580), and potencies were calculated relative to both the existing US Standard (lot J) and the IS (89/588). Data analysis of a total of 128 assays indicated that sample D would make an ideal replacement joint Standard with a potency of 110 IU/ampoule (equivalent to 110 US units per ampoule) based on data from clotting assays. No significant differences in results were observed using fibrinogen of human or bovine origin, or using human plasma. Comparisons of chromogenic and clotting assays indicated that sample D had a similar high proportion of alpha thrombin to the current IS for Alpha Thrombin (89/588). Sample D was adopted as the IS for Thrombin (01/580) and the US Standard (lot K) with a potency of 110 IU/ampoule.     

Lentiviral transduction of murine oligodendrocytes in vivo

Lentiviral vectors are used widely to direct efficient gene transfer in vivo. We examined cell-specific expression in adult murine white matter after stereotaxic microinjection of four lentiviral constructs. We synthesized vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviruses with combinations of two promoters, cytomegalovirus (CMV) or myelin basic protein (MBP), and two reporter sequences, cytosolic enhanced green fluorescent protein (eGFP) or a plasma membrane-targeted eGFP (human lymphocyte-specific protein tyrosine kinase [Lck]-eGFP). For all constructs, intracerebral injections to lateral corpus callosum resulted in widespread GFP expression in forebrain white matter glial cells. Intense cellular GFP fluorescence was observed within 3 days after injection and lasted for at least 28 days. The CMV promoter directed GFP expression in multiple glial cell types, whereas the MBP promoter targeted GFP specifically to oligodendrocytes. Expression of the membrane-targeted Lck-eGFP construct distinctly labeled individual myelinating processes of oligodendrocytes. Lentiviral constructs expressing eGFP or Lck-eGFP under the MBP promoter provide excellent visualization of oligodendrocyte morphology in intact white matter, and may prove valuable for delivering additional genes of interest to oligodendrocytes in vivo.     

Vascular access monitoring improves outcomes

Vascular access monitoring can identify patients at increased risk of future access thrombosis. When coupled with a program of elective stenosis correction, access thrombosis rates decline approximately 50-75%. This results in arteriovenous (AV) fistula thrombosis rates of 0.1-0.2/patient year (vs. 0.2-0.4 at baseline) and AV graft thrombosis rates <0.5/patient year (vs. 0.8-1.2 thromboses/patient year at baseline). Evaluating the long-term impact on access survival remains problematic. There are no large-scale randomized trials and existing studies exhibit marked differences in target populations, clinical protocols and outcome definitions. Differences in payment systems also significantly influence the efficacy of monitoring and intervention programs. Despite these challenges, the current data support the K/DOQI recommendations that all patients undergo a program of regular access monitoring preferably by access flow measurement coupled with prompt imaging and elective stenosis correction for low flow accesses.     

Lisfranc injuries

Lisfranc/midfoot injuries are complex injuries that are frequently overlooked. The consequences of missing these injuries can lead to long-term deformity and disability. Timely diagnosis, whether by primary or later treating physicians, can ensure this does not happen. Proper physical examination and imaging studies allow the physician to find these injuries and determine if open treatment is required. Open treatment can be approached and performed in a number of ways, depending on geographical preference. However, one concept that remains across all the various techniques is perfect anatomical reduction before the fixation technique is applied. Intraoperative and postoperative variations are all secondary concerns after anatomical reduction.     

Collaborative study for establishment of a global standard for the potency assay of human anti-D immunoglobulin

An international collaborative study aimed at establishing a global standard for the potency assay of anti-D immunoglobulin was started in 2002. 25 laboratories participated in this study run under the common aegis of the World Health Organization, the United States Food and Drug Administration (US-FDA) and the European Directorate for the Quality of Medicines (EDQM). The potencies of three candidate materials and the US-FDA standard (lot 3) included for comparison were evaluated using AutoAnalyzer, competitive enzyme-linked immunoassay (competitive EIA), flow cytometric methods or own "in-house" methods. Critical reagent, standardised procedures and standardised assay design were provided for either method, where appropriate. Central statistical evaluation of the potency data submitted by the participants was performed using a parallel line model. Agreement between laboratories and assay methods for all samples was observed. Intra-laboratory variability was lowest for laboratories performing flow cytometry and highest for laboratories that performed their in-house methods. Inter-laboratory variability was acceptable for all samples when assayed by AutoAnalyzer, competitive (EIA) and flow cytometric methods. It was concluded that sample A is most suitable to serve as a global standard and that sample C could serve as a reserve European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) batch provided that suitable stability is demonstrated. Sample A was adopted by the Ph. Eur. Commission at its 115th session (March 2003) as the first Ph. Eur. BRP (available from the EDQM: catalog number Y0000219) with the assigned potency of 285 IU/ampoule.     

Adeno-associated virus 2-mediated gene therapy decreases autofluorescent storage material and increases brain mass in a murine model of infantile neuronal ceroid lipofuscinosis

Infantile neuronal ceroid lipofuscinosis (INCL) is the earliest onset form of a class of inherited neurodegenerative disease called Batten disease. INCL is caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). Autofluorescent storage material accumulates in virtually all tissues in INCL patients, including the brain, and leads to widespread neuronal loss and cortical atrophy. To determine the efficacy of viral-mediated gene therapy, we injected a recombinant adeno-associated virus 2 vector encoding human PPT1 (rAAV-PPT1) intracranially (I.C.) into a murine model of INCL. INCL mice given four I.C. injections of rAAV-PPT1 as newborns exhibited PPT1 activity near the injection sites and decreased secondary elevations of another lysosomal enzyme. In addition, storage material was decreased in cortical, hippocampal, and cerebellar neurons, and brain weights and cortical thicknesses were increased. These data demonstrate that an adeno-associated virus 2 (AAV2)-mediated gene therapy approach may provide some therapeutic benefit for INCL.     

VEGF disrupts the neonatal blood-brain barrier and increases life span after non-ablative BMT in a murine model of congenital neurodegeneration caused by a lysosomal enzyme deficiency

The course of certain congenital neurodegenerative diseases like lysosomal storage diseases (LSDs) begins shortly after birth and can progress quickly. Ideally, therapeutic interventions for LSDs, which include bone marrow transplantation (BMT), recombinant enzyme replacement, or systemic viral-mediated gene therapy, should be initiated at birth. However, the blood-brain barrier (BBB) remains an obstacle to effective therapy even when these strategies are initiated at birth. We studied whether VEGF, an endothelial cell mitogen and permeability factor, can open the BBB in newborn mice for therapeutic purposes. Intravenous (IV) administration of VEGF at birth increased BBB permeability within 2 h. The increased permeability persisted for at least 24 h, became undetectable 48 h after injection, and was restricted to newborns. Systemic VEGF treatment before BMT or administration of recombinant lentivirus resulted in increased numbers of both donor cells and virus-transduced cells, respectively, in the recipient brain. Administration of VEGF before BMT in newborn mice with a neurodegenerative LSD, globoid-cell leukodystrophy, resulted in a significant increase in life span compared to affected animals that were injected with saline before BMT.     

Microscopic imaging of extended tissue volumes

1. Detailed information about three-dimensional structure is key to understanding biological function. 2. Confocal laser microscopy has made it possible to reconstruct three-dimensional organization with exquisite resolution at cellular and subcellular levels. 3. There have been few attempts to acquire large image volumes using the confocal laser scanning microscope. 4. Previously, we have used manual techniques to construct extended volumes (several mm in extent, at 1.5 microm voxel size) of myocardial tissue. 5. We are now developing equipment and efficient automated methods for acquiring extended morphometric databases using confocal laser scanning microscopy.     

Perception of health status by homeless US veterans

Perceptions of health status among 331 homeless veterans and homeless nonveterans were examined. Homeless veterans were significantly less apt to perceive their health as fair/poor (8%) compared to non-veteran homeless men (19%). Homeless veterans were also more likely to report having a regular source of care (57% versus 36%). Logistic regression analysis indicated the adjusted odds of fair/poor health were more than two times greater for persons reporting depressive symptomatology than for those without this history; veterans continue to remain less likely to report fair/poor health than nonveterans. High rates of substance abuse were observed for the entire sample. Such differences in perceived health result in important health access issues.     

Hospital response to chemical terrorism: personal protective equipment, training, and operations planning

BACKGROUND: Hospitals distant from the immediate site of an incident involving a hazardous materials (HAZMATs) release which could include chemical warfare agents, must develop emergency response plans (ERPs) to protect healthcare professionals if they receive potentially contaminated victims. The ERP must address OSHA, EPA, and JCAHO requirements. METHODS: The VHA convened groups to develop a hazard and exposure assessment, identify actions for compliance with existing regulatory standards, and review site and operational planning issues. Exposure modeling results were used to derive relationships between operational parameters (time and distance from sites/sources) and potential exposure for healthcare workers. RESULTS: According to exposure modeling, level C personal protective equipment is adequate to protect hospital staff distant from the chemical release site. Decontamination runoff and contaminated clothing should also be controlled to limit exposure. CONCLUSIONS: Development and coordination of ERPs must include the local emergency planning committee, with clear assignment of tasks, locations, and training in order to prevent exposures to healthcare workers.