A Comparison of Red Fluorescent Proteins to Model DNA Vaccine Expression by Whole Animal In Vivo Imaging

DNA vaccines can be manufactured cheaply, easily and rapidly and have performed well in pre-clinical animal studies. However, clinical trials have so far been disappointing, failing to evoke a strong immune response, possibly due to poor antigen expression. To improve antigen expression, improved technology to monitor DNA vaccine transfection efficiency is required. In the current study, we compared plasmid encoded tdTomato, mCherry, Katushka, tdKatushka2 and luciferase as reporter proteins for whole animal in vivo imaging. The intramuscular, subcutaneous and tattooing routes were compared and electroporation was used to enhance expression. We observed that overall, fluorescent proteins were not a good tool to assess expression from DNA plasmids, with a highly heterogeneous response between animals. Of the proteins used, intramuscular delivery of DNA encoding either tdTomato or luciferase gave the clearest signal, with some Katushka and tdKatushka2 signal observed. Subcutaneous delivery was weakly visible and nothing was observed following DNA tattooing. DNA encoding haemagglutinin was used to determine whether immune responses mirrored visible expression levels. A protective immune response against H1N1 influenza was induced by all routes, even after a single dose of DNA, though qualitative differences were observed, with tattooing leading to high antibody responses and subcutaneous DNA leading to high CD8 responses. We conclude that of the reporter proteins used, expression from DNA plasmids can best be assessed using tdTomato or luciferase. But, the disconnect between visible expression level and immunogenicity suggests that in vivo whole animal imaging of fluorescent proteins has limited utility for predicting DNA vaccine efficacy

Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein

A method for non-invasive visualization of genetically labelled cells in animal disease models with micron-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the “optical window” above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune, previously the brightest monomeric FP when excited beyond 600 nm. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence, while the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts and stem cells into myocytes in living mice with high anatomical detail

Risk factors for infections caused by carbapenem-resistant Enterobacterales: an international matched case-control-control study (EURECA)

Cases were patients with complicated urinary tract infection (cUTI), complicated intraabdominal (cIAI), pneumonia or bacteraemia from other sources (BSI-OS) due to CRE; control groups were patients with infection caused by carbapenem-susceptible Enterobacterales (CSE), and by non-infected patients, respectively. Matching criteria included type of infection for CSE group, ward and duration of hospital admission. Conditional logistic regression was used to identify risk factors. Findings Overall, 235 CRE case patients, 235 CSE controls and 705 non-infected controls were included. The CRE infections were cUTI (133, 56.7%), pneumonia (44, 18.7%), cIAI and BSI-OS (29, 12.3% each). Carbapenemase genes were found in 228 isolates: OXA-48/like, 112 (47.6%), KPC, 84 (35.7%), and metallo-beta-lactamases, 44 (18.7%); 13 produced two. The risk factors for CRE infection in both type of controls were (adjusted OR for CSE controls; 95% CI; p value) previous colonisation/infection by CRE (6.94; 2.74-15.53; <0.001), urinary catheter (1.78; 1.03-3.07; 0.038) and exposure to broad spectrum antibiotics, as categorical (2.20; 1.25-3.88; 0.006) and time-dependent (1.04 per day; 1.00-1.07; 0.014); chronic renal failure (2.81; 1.40-5.64; 0.004) and admission from home (0.44; 0.23-0.85; 0.014) were significant only for CSE controls. Subgroup analyses provided similar results. Interpretation The main risk factors for CRE infections in hospitals with high incidence included previous coloni-zation, urinary catheter and exposure to broad spectrum antibiotics

Ex vivo platelets' aggregation and von Willebrand factors in type I diabetes mellitus

Object of this study was the investigation of the effect of type I diabetes mellitus on the platelet count and platelet functionality, as well as, on the levels of von Willebrand (vWF) and VIII: C factors. Ninety four children with diabetes (47 males and 47 females) and 52 (21 males and 31 females) healthy children aged 2-24 years were studied following parental consent. The children with diabetes were followed in the Pediatric Department’s Diabetes Centre, Faculty of Nursing, University of Athens. The healthy children were selected as controls from children admitted to the “P & A Kyriakou” Children’s Hospital for clinical - laboratory tests and minor surgery. The laboratory tests were carried out at the Laboratory of Hygiene and Epidemiology of the University of Athens Medical School and the Hematology Department of the “P & A Kyriakou” Children’s Hospital in Athens. Platelet aggregation was measured with an aggregometer PAP – 4 model 430, Biodata Co, using a turbidometric technique, according to the method of Born and O’Brien, by the addition of three agonists (ADP, collagen, ristocetine). Platelet count and size were determined with the Coulter STKS hematologic analyzer. The plasma antigenic activity of vWF was measured by Laurell electroimmunodifusion. The vWF ristocetine cofactor was measured after the platelets were washed and fixed. The concentration of VIII: C factor was measured with the method of APTT in real time. The tests (fibrinogen, glycosylated Hb) were carried out with routine laboratory techniques. Analysis of data was performed using the SPSS statistical - analysis package. The outcome (or dependent variable) was: a) the platelet count, b) the platelet aggregation ex vivo after adding three different agonists (ADP, collagen, ristocetine), c) the vWF και VIII: C factors. As predictor (or independent) variable was the existence or none of type I diabetes mellitus. The confounders which were controlled in data analysis were: a) the age, b) the gender, c) the system of ABO blood group, d) the height, e) the weight, f) the glycosylated Hb και g) the duration of diabetes. The following results were obtained: 1) A smaller number of platelets in children with diabetes (x±SE = 240.000 ± 6.000/mm3) was observed as compared with the healthy ones (x±SE = 288.000 ± 10.000/mm3) 2) No statistically significant difference was found in the functionality of the platelets (in vitro aggregability to ADP, collagen and ristocetine) between children with diabetes and healthy individuals 3) No differences were observed in the distribution of children with diabetes and healthy children among the four groups of the ABO system 4) Greater antigenic activity of the vWF was observed in children with diabetes (95% CI of β = 19,3 - 45) as compared to healthy children. The same positive relation was observed regarding the vWF Rcof (95% CI of β = 28,2 - 57,9) 5) Higher levels of the factor VIII: C were found in children with diabetes (95% CI of β = 5,34 - 43,5) as compared to healthy children 6) The level of glycosylated Hb in children with diabetes depends positively on the duration of the disease and gender.Μελετήθηκε η επίδραση του ινσουλινοεξαρτώμενου σακχαρώδη διαβήτη (ΙΕΣΔ) στον αριθμό και τη λειτουργικότητα των αιμοπεταλίων ex vivo, στα επίπεδα του vW (αντιγόνου και συμπαράγοντα ριστοσετίνης), του παράγοντα VΙΙΙ: C και της γλυκοζιωμένης αιμοσφαιρίνης. Στην εργασία συμμετείχαν, μετά από έγγραφη συναίνεση των γονέων τους, 94 διαβητικά παιδιά (47 αγόρια και 47 κορίτσια) και 52 υγιή παιδιά (21 αγόρια και 31 κορίτσια), ηλικίας 2-24 ετών. Τα διαβητικά παιδιά παρακολουθούνταν από το Διαβητολογικό Κέντρο του ΠΓΝΑ «Π&Α ΚΥΡΙΑΚΟΥ». Οι εργαστηριακές δοκιμασίες γινόντουσαν στα ερευνητικά εργαστήρια της Υγιεινής και Επιδημιολογίας του Πανεπιστημίου Αθηνών και του Αιματολογικού Εργαστηρίου του ΠΓΝΑ «Π&Α ΚΥΡΙΑΚΟΥ». Για τη μελέτη της συγκολλητικότητας των αιμοπεταλίων ex vivo, κατόπιν προσθήκης τριών αγωνιστών (ADP, κολλαγόνο, ριστοσετίνη) χρησιμοποιήθηκε το συγκολλητινόμετρο (αγγρεγκόμετρο) ΡΑΡ - 4 model 430 της BioData. Για την αρίθμηση των αιμοπεταλίων χρησιμοποιήθηκε ο αιματολογικός αναλυτής Coulter STKS. Η ανάλυση των δεδομένων έγινε σε ηλεκτρονικό υπολογιστή με το στατιστικό πακέτο SPSS. Εφαρμόστηκε το υπόδειγμα της απλής και πολλαπλής γραμμικής παλινδρόμησης και εξουδετερώθηκαν οκτώ συγχυτικοί παράγοντες (ηλικία, φύλο, ομάδα αίματος ΑΒΟ, ινωδογόνο, ανάστημα, σωματικό βάρος, γλυκοζιωμένη αιμοσφαιρίνη, χρονικό διάστημα από την εμφάνιση του σακχαρώδη διαβήτη). Τα αποτελέσματα συνοψίζονται ως κάτωθι: 1. Διαπιστώθηκε μικρότερος αριθμός αιμοπεταλίων στα διαβητικά παιδιά (x±SE = 240.000 ± 6.000/mm3) σε σχέση με τα υγιή (x±SE = 288.000 ± 10.000/mm3). 2. Αντίθετα δεν διαπιστώθηκε, στατιστικά σημαντική, μεταβολή της λειτουργικότητας των αιμοπεταλίων στα διαβητικά παιδιά, όπως αυτή μελετάται in vitro με τη συγκολλητικότητα των αιμοπεταλίων κατόπιν προσθήκης τριών αγωνιστών: ADP (95% ΔΕ του β = -8,8 έως +3,2), κολλαγόνο (95% ΔΕ του β = - 2,18 έως +4,49), ριστοσετίνη (95% ΔΕ του β = - 44,85 έως +7,86). 3. Δεν παρατηρήθηκε επίσης διαφορά στην κατανομή των διαβητικών και υγιών παιδιών στις τέσσερις ομάδες αίματος του συστήματος ΑΒΟ. 4. Διαπιστώθηκαν μεγαλύτερες συγκεντρώσεις του αντιγόνου του παράγοντα vW στα διαβητικά (95% ΔΕ του β = 19,3 έως 45) σε σχέση με τα υγιή παιδιά και υψηλότερες τιμές του vWRCoF (95% ΔΕ του β = 23,2 έως 57,9). 5. Υψηλότερα επίπεδα του παράγοντα VIII: C βρέθηκαν στα διαβητικά σε σχέση με τα υγιή παιδιά (95% ΔΕ του β = 5,34 έως 43,5). 6. Τα επίπεδα της γλυκοζιωμένης αιμοσφαιρίνης στα διαβητικά παιδιά εξαρτώνται όχι μόνο από τη διάρκεια της νόσου αλλά και από το φύλο

Colistin susceptibility testing by Etest and disk diffusion methods

The accuracy of disk susceptibility methods for colistin against 778 bacterial pathogens was evaluated in comparison with Etest using interpretive criteria available from the Clinical and Laboratory Standards Institute (CLSI). Colistin exhibited excellent activity against Acinetobacter baumannii and Escherichia coli isolates (minimum inhibitory concentration for 90% of the organisms (MIC90) = 0.5 mg/L), whilst it was less active both against Enterobacter spp. and Klebsiella pneumoniae (MIC for 50% of the organisms (MIC50) = 0.5 mg/L, MIC90 = 16 mg/L). Colistin also showed good activity against Pseudomonas aeruginosa (MIC90 = 2 mg/L, MIC50 = 1 mg/L) but poor activity against Stenotrophomonas maltophilia (MIC50 = 8 mg/L, MIC90 = 128 mg/L). Only 0.8% of minor errors were observed between the studied methods for P. aeruginosa isolates when the CLSI criteria were applied. All A. baumannii isolates with a zone diameter &lt;= 12mm were resistant and those with a zone diameter &gt;= 14mm were susceptible according to MIC breakpoints established by the CLSI. Among nine isolates exhibiting a zone diameter of 13 mm, one was resistant to colistin (MIC = 8 mg/L) and eight isolates were susceptible (MIC = 0.5 mg/L). Applying a MIC breakpoint of &lt;= 2 mg/L for susceptibility in Enterobacteriaceae, all isolates with a zone diameter &gt;= 14mm were susceptible, whilst all isolates with a zone diameter &lt;= 11mm were resistant. Among isolates with zone diameters of 12-13 mm, 59% were characterised as susceptible. Major errors were observed only in K. pneumoniae isolates at a rate of 0.8%. The poor agar diffusion characteristics of colistin limit the predictive accuracy of the disk diffusion test and consequently values of 12-13mm should be confirmed with MIC determination by Etest or broth dilution method. (C) 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved

Infective Endocarditis in Patients on Chronic Hemodialysis

International audienceInfective endocarditis (IE) is a common and serious complication in patients receiving chronic hemodialysis (HD)