Quality-Controlled Small-Scale Production of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials

We describe the small-scale, laboratory-based, production and quality control of a cocktail, consisting of exclusively lytic bacteriophages, designed for the treatment of Pseudomonas aeruginosa and Staphylococcus aureus infections in burn wound patients. Based on succesive selection rounds three bacteriophages were retained from an initial pool of 82 P. aeruginosa and 8 S. aureus bacteriophages, specific for prevalent P. aeruginosa and S. aureus strains in the Burn Centre of the Queen Astrid Military Hospital in Brussels, Belgium. This cocktail, consisting of P. aeruginosa phages 14/1 (Myoviridae) and PNM (Podoviridae) and S. aureus phage ISP (Myoviridae) was produced and purified of endotoxin. Quality control included Stability (shelf life), determination of pyrogenicity, sterility and cytotoxicity, confirmation of the absence of temperate bacteriophages and transmission electron microscopy-based confirmation of the presence of the expected virion morphologic particles as well as of their specific interaction with the target bacteria. Bacteriophage genome and proteome analysis confirmed the lytic nature of the bacteriophages, the absence of toxin-coding genes and showed that the selected phages 14/1, PNM and ISP are close relatives of respectively F8, φKMV and phage G1. The bacteriophage cocktail is currently being evaluated in a pilot clinical study cleared by a leading Medical Ethical Committee

Awareness of dental practitioners to cope with a medical emergency: a survey in Belgium

Objective: The aim of the study is to determine whether in Belgium dentists feel confident to diagnose a medical emergency situation in their dental practice. Methods: A questionnaire was completed by 7.0% of the active Belgian general dental practitioners (n=548) including questions on the frequency and knowledge of medical emergency situations in the dental office, history of dental training to treat emergency situations and confidence level in treating emergency situations. Results: A medical history of each patient was taken by 55.3% of the dentists. A link was found between years since graduation and the systematic decline of a medical history in a new patient (P=0.001): the older the dentist, the less consistent was the updating of medical history. Almost 50% of the dentists (49.4%) never participated in any basic life support (BLS) training during their undergraduate education. Moreover, 78.3% never had any paediatric BLS training during undergraduate education and BLS training after graduation was lacking by 37.2% of the dentists. Conclusion: Knowledge of BLS should be fundamental to medical professionals. The more BLS training a practitioner has experienced, the more self-secure they feel coping with an emergency situation

Determination of relative amounts of inositol trisphosphate receptor mRNA isoforms by ratio polymerase chain reaction

The relative expression of different inositol 1,4,5-trisphosphate receptor (InsP3R) mRNA was determined in a selection of murine and rat cell types that are commonly used to study InsP3-mediated Ca2+ signaling. Different mRNA species (encoding the known InsP3R isoforms) were co-amplified using common polymerase chain reaction primer pairs that recognized sequences that are totally conserved between the various InsP3R. Specific identification of the co-amplified sequences was done by restriction site analysis. In cerebellum, mRNA encoding InsP3R-I accounted for > 90% of the total InsP3R mRNA. This isoform was also present in all other cell types tested and was often the major isoform. In contrast, the level of expression of the other isoforms was cell type-specific. A new InsP3R isoform (type V) was detected that had 94.5% sequence identity with the InsP3R-II in the amplified region. Interestingly, this isoform was largely expressed in murine but not in rat cells. We functionally characterized InsP3R-V using the mouse fibroblast C3H10T½ cells, where mRNA encoding InsP3R-V accounted for 76.4% of the total InsP3R mRNA. InsP3-induced Ca2+ release in permeabilized C3H10T½ cells was regulated by luminal and cytosolic Ca2+, stimulated by thimerosal, and inhibited by caffeine

Does cruciate retention primary total knee arthroplasty affect proprioception, strength and clinical outcome?

It remains unclear what the contribution of the PCL is in total knee arthroplasty (TKA). The goal of this study was to investigate the influence of the PCL in TKA in relationship to clinical outcome, strength and proprioception. Two arthroplasty designs were compared: a posterior cruciate-substituting (PS) and a posterior cruciate-retaining (CR) TKA. A retrospective analysis was performed of 27 CR and 18 PS implants with a minimum of 1 year in vivo. Both groups were compared in terms of clinical outcome (range of motion, visual analogue scale for pain, Hospital for Special Surgery Knee Scoring system, Lysholm score and Knee Injury and Osteoarthritis Outcome Score), strength (Biodex System 3 Dynamometer(A (R))) and proprioception (balance and postural control using the Balance Master system(A (R))). Each design was also compared to the non-operated contralateral side in terms of strength and proprioception. There were no significant differences between both designs in terms of clinical outcome and strength. In terms of proprioception, only the rhythmic weight test at slow and moderate speed shifting from left to right was significant in favour of the CR design. None of the unilateral stance tests showed any significant difference between both designs. There was no difference in terms of strength and proprioception between the operated side and the non-operated side. Retaining the PCL in TKA does not result in an improved performance in terms of clinical outcome and proprioception and does not show any difference in muscle strength

Inositol trisphosphate producing agonists do not mobilize the thapsigargin-insensitive part of the endoplasmic-reticulum and Golgi Ca2+ store

Non-mitochondrial intracellular Ca2+ stores contain both thapsigargin-sensitive sarco(endo)plasmic-reticulum Ca2+-ATPases (SERCA) and thapsigargin-insensitive secretory-pathway Ca2+-ATPases (SPCA1). We now have studied the Ca2+-release properties of the compartments associated with these pumps in intact, i.e. non-permeabilized, cells of different origin (HeLa, keratinocytes, 16HBE14o-, COS-1, A7r5) and with different approaches (45Ca2+ fluxes, Ca2+ imaging and measurements of the free luminal [Ca2+] in the endoplasmic-reticulum and the Golgi apparatus using targeted aequorin). Application of an extracellular agonist in the absence of thapsigargin induced in all cells a Ca2+ release from both the endoplasmic-reticulum and the Golgi apparatus. The agonists were not able to release Ca2+ in the presence of 10 microM thapsigargin, except in COS-1 cells overexpressing SPCA1, where this pump not only appeared in the Golgi compartment but also overflowed into the agonist-sensitive part of the endoplasmic-reticulum. We conclude that the subcompartments of the endoplasmic-reticulum and of the Golgi complex that endogenously express SPCA1 are insensitive to agonist stimulation.status: publishe

The Ca2+/Mn2+ pumps in the Golgi apparatus

Recent evidence highlights the functional importance of the Golgi apparatus as an agonist-sensitive intracellular Ca(2+) store. Besides Ca(2+)-release channels and Ca(2+)-binding proteins, the Golgi complex contains Ca(2+)-uptake mechanisms consisting of the well-known sarco/endoplasmic reticulum Ca(2+)-transport ATPases (SERCA) and the much less characterized secretory-pathway Ca(2+)-transport ATPases (SPCA). SPCA supplies the Golgi compartments and, possibly, the more distal compartments of the secretory pathway with both Ca(2+) and Mn(2+) and, therefore, plays an important role in the cytosolic and intra-Golgi Ca(2+) and Mn(2+) homeostasis. Mutations in the human gene encoding the SPCA1 pump (ATP2C1) resulting in Hailey-Hailey disease, an autosomal dominant skin disorder, are discussed.status: publishe

SPCA1 pumps and Hailey-Hailey disease

Both the endoplasmic reticulum and the Golgi apparatus are agonist-sensitive intracellular Ca2+ stores. The Golgi apparatus has Ca2+-release channels and a Ca2+-uptake mechanism consisting of sarco(endo)plasmic-reticulum Ca2+-ATPases (SERCA) and secretory-pathway Ca2+-ATPases (SPCA). SPCA1 has been shown to transport both Ca2+ and Mn2+ in the Golgi lumen and therefore plays an important role in the cytosolic and intra-Golgi Ca2+ and Mn2+ homeostasis. Human genetic studies have provided new information on the physiological role of SPCA1. Loss of one functional copy of the SPCA1 (ATP2C1) gene causes Hailey-Hailey disease, a skin disorder arising in the adult age with recurrent vesicles and erosions in the flexural areas. Here, we review recent experimental evidence showing that the Golgi apparatus plays a much more important role in intracellular ion homeostasis than previously anticipated.status: publishe