Microneedle devices have been proposed as a minimally invasive delivery system for the intradermal administration of nucleic acids, both plasmid DNA (pDNA) and siRNA, to treat localised disease or ...provide vaccination. Different microneedle types and application methods have been investigated in the laboratory, but limited and irreproducible levels of gene expression have proven to be significant challenges to pre-clinical to clinical progression. This study is the first to explore the potential of a hollow microneedle device for the delivery and subsequent expression of pDNA in human skin. The regulatory approved MicronJet600® (MicronJet hereafter) device was used to deliver reporter plasmids (pCMVβ and pEGFP-N1) into viable excised human skin. Exogenous gene expression was subsequently detected at multiple locations that were distant from the injection site but within the confines of the bleb created by the intradermal bolus. The observed levels of gene expression in the tissue are at least comparable to that achieved by the most invasive microneedle application methods e.g. lateral application of a microneedle. Gene expression was predominantly located in the epidermis, although also evident in the papillary dermis. Optical coherence tomography permitted real time visualisation of the sub-surface skin architecture and, unlike a conventional intradermal injection, MicronJet administration of a 50μL bolus appears to create multiple superficial microdisruptions in the papillary dermis and epidermis. These were co-localised with expression of the pCMVβ reporter plasmid. We have therefore shown, for the first time, that a hollow microneedle device can facilitate efficient and reproducible gene expression of exogenous naked pDNA in human skin using volumes that are considered to be standard for intradermal administration, and postulate a hydrodynamic effect as the mechanism of gene delivery.
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Antigen specific immunotherapy aims to tolerise patients to specific autoantigens that are responsible for the pathology of an autoimmune disease. Immune tolerance is generated in ...conditions where the immune response is suppressed and thus gold nanoparticles (AuNPs) are an attractive drug delivery platform due to their anti-inflammatory effects and their potential to facilitate temporal and spatial delivery of a peptide autoantigen in conjunction with pro-tolerogenic elements. In this study we have covalently attached an autoantigen, currently under clinical evaluation for the treatment of type 1 diabetes (PIC19-A3 peptide), to AuNPs to create nanoscale (<5 nm), negatively charged (−40 to −60 mV) AuNP-peptide complexes for immunotherapy. We also employ a clinically approved microneedle delivery system, MicronJet600, to facilitate minimally-invasive intradermal delivery of the nanoparticle constructs to target skin-resident antigen presenting cells, which are known to be apposite target cells for immunotherapy. The AuNP-peptide complexes remain physically stable upon extrusion through microneedles and when delivered into ex vivo human skin they are able to diffuse rapidly and widely throughout the dermis (their site of deposition) and, perhaps more surprisingly, the overlying epidermal layer. Intracellular uptake was extensive, with Langerhans cells proving to be the most efficient cells at internalising the AuNP-peptide complex (94% of the local population within the treated region of skin). In vitro studies showed that uptake of the AuNP-peptide complexes by dendritic cells reduced the capacity of these cells to activate naïve T cells. This indicator of biological functionality encourages further development of the AuNP-peptide formulation, which is now being evaluated in clinical trials.
The full-field stimulus test (FST) is a psychophysical technique designed for the measurement of visual function in low vision. The method involves the use of a ganzfeld stimulator, as used in ...routine full-field electroretinography, to deliver full-field flashes of light. This guideline was developed jointly by the International Society for Clinical Electrophysiology of Vision (ISCEV) and Imaging and Perimetry Society (IPS) in order to provide technical information, promote consistency of testing and reporting, and encourage convergence of methods for FST. It is intended to aid practitioners and guide the formulation of FST protocols, with a view to future standardisation.
Background. Klebsiella pneumoniae isolates harboring the K. pneumoniae carbapenemase gene (bla KPC ) are creating a significant healthcare threat in both acute and long-term care facilities (LTCFs). ...As part of a study conducted in 2004 to determine the risk of stool colonization with extended-spectrum cephalosporin-resistant gram-negative bacteria, 12 isolates of K. pneumoniae that exhibited nonsusceptibility to extended-spectrum cephalosporins were detected. All were gastrointestinal carriage isolates that were not associated with infection. Methods. Reassessment of the carbapenem minimum inhibitory concentrations using revised 2011 Clinical Laboratory Standards Institute breakpoints uncovered carbapenem resistance. To further investigate, a DNA microarray assay, PCR-sequencing of bla genes, immunoblotting, repetitive-sequence-based PCR (rep-PCR) and multilocus sequence typing (MLST) were performed. Results. The DNA microarray detected bla KPC in all 12 isolates, and bla KPC-3 was identified by PCR amplification and sequencing of the amplicon. In addition, a bla SHV-11 gene was detected in all isolates. Immunoblotting revealed "low-level" production of the K. pneumoniae carbapenemase, and rep-PCR indicated that all bla KPC-3 -positive K. pneumoniae strains were genetically related (≥98% similar). According to MLST, all isolates belonged to sequence type 36. This sequence type has not been previously linked with bla KPC carriage. Plasmids from 3 representative isolates readily transferred the bla KPC-3 to Escherichia coli J-53 recipients. Conclusions. Our findings reveal the "silent" dissemination of bla KPC-3 as part of Tn4401b on a mobile plasmid in Northeast Ohio nearly a decade ago and establish the first report, to our knowledge, of K. pneumoniae containing bla KPC-3 in an LTCF caring for neurologically impaired children and young adults.
Abstract
Antigen-specific immunotherapy is an immunomodulatory strategy for autoimmune diseases, such as type 1 diabetes, in which patients are treated with autoantigens to promote immune tolerance, ...stop autoimmune β-cell destruction and prevent permanent dependence on exogenous insulin. In this study, human proinsulin peptide C19-A3 (known for its positive safety profile) was conjugated to ultrasmall gold nanoparticles (GNPs), an attractive drug delivery platform due to the potential anti-inflammatory properties of gold. We hypothesised that microneedle intradermal delivery of C19-A3 GNP may improve peptide pharmacokinetics and induce tolerogenic immunomodulation and proceeded to evaluate its safety and feasibility in a first-in-human trial. Allowing for the limitation of the small number of participants, intradermal administration of C19-A3 GNP appears safe and well tolerated in participants with type 1 diabetes. The associated prolonged skin retention of C19-A3 GNP after intradermal administration offers a number of possibilities to enhance its tolerogenic potential, which should be explored in future studies
Background Resistance to carbapenems among Acinetobacter baumannii and Klebsiella pneumoniae presents a serious therapeutic and infection control challenge. We describe the epidemiology and genetic ...basis of carbapenem resistance in A. baumannii and K. pneumoniae in a six-hospital healthcare system in Northeast Ohio. Methods Clinical isolates of A. baumannii and K. pneumoniae distributed across the healthcare system were collected from April 2007 to April 2008. Antimicrobial susceptibility testing was performed followed by molecular analysis of carbapenemase genes. Genetic relatedness of isolates was established with repetitive sequence-based PCR (rep-PCR), multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) and PFGE. Clinical characteristics and outcomes of patients were reviewed. Results Among 39 isolates of A. baumannii, two predominant genotypes related to European clone II were found. Eighteen isolates contained blaOXA-23, and four isolates possessed blaOXA-24/40. Among 29 K. pneumoniae isolates with decreased susceptibility to carbapenems, two distinct genotypes containing blaKPC-2 or blaKPC-3 were found. Patients with carbapenem-resistant A. baumannii and K. pneumoniae were elderly, possessed multiple co-morbidities, were frequently admitted from and discharged to post-acute care facilities, and experienced prolonged hospital stays (up to 25 days) with a high mortality rate (up to 35%). Conclusion In this outbreak of carbapenem-resistant A. baumannii and K. pneumoniae across a healthcare system, we illustrate the important role post-acute care facilities play in the dissemination of multidrug-resistant phenotypes.
A daily dose of vitamin K antagonists (VKAs) may vary and its range depends on various interrelated factors. Low responsiveness to VKA (defined as a failure to achieve a target international ...normalized ratio INR) is associated with polymorphisms of the vitamin K epoxide reductase-oxidase complex gene (
VKORC1
). A highly prevalent promoter single-nucleotide polymorphism (VKORC1−1639 G>A, rs 17878363) impairs
VKORC1
expression and determines the interindividual variability of the target INR. We studied 57 patients receiving oral anticoagulation, including 50 subjects treated with acenocoumarol (mean dose: 5.7±2.3 mg/day) and 7 treated with warfarin (mean dose: 9.6±4.2 mg/day). The indications for the use of oral anticoagulant therapy were as follows: deep-vein thrombosis (
N
= 23); pulmonary embolism (
N
= 20); arterial thrombosis (
N
= 5); stroke (
N
= 4); atrial fibrillation with transient ischemic attacks (
N
= 2), and history of multiple thromboembolic events (
N
= 3). Identification of the
VKORC1
genomic variation was performed using DNA sequencing methods. The prevalence of the mutated allele (
VKORC1
-1639A) was 41%. The
VKORC1
-1639G allele carriers required a higher daily dose of acenocoumarol (5.9±1.9 mg) than the noncarriers (4.1±3.3 mg;
P <
0.001). All of 5 low responders (who failed to achieve a target INR using standard dose requirements of VKAs) were homozygous for the 1639G allele. Low responders did not differ from good responders with respect to age, gender, and body mass index. Our findings suggest the potential benefits from pharmacogenetic testing, and provide evidence that the
VKORC1
−1639 G>A gene polymorphism may explain at least in part the low responsiveness to acenocoumarol.
Gram-negative organisms that are resistant to parenteral antibiotics are a growing threat to hospitalized patients. This study was conducted to define the epidemiologic characteristics of these ...organisms during a nonoutbreak period in a neonatal intensive care unit (NICU).
Nasopharyngeal and rectal swab specimens were obtained 3 times a week from every infant in a tertiary care NICU during a 12-month period. Specimens were processed to identify aerobic Gram-negative species resistant to gentamicin, piperacillin-tazobactam, or ceftazidime. Selected clinical parameters were tested for their association with colonization with a resistant organism. Restriction endonuclease digests of genomic DNA were derived from isolates of the most frequently occurring species. The fragments were analyzed by pulsed-field gel electrophoresis (PFGE) to determine the genetic relatedness of the various isolates and thereby determine the length of colonization, the frequency of horizontal transmission, and the size and duration of clusters.
A total of 101 infants (8.6%) of 1180 admissions were colonized with at least 1 antibiotic-resistant bacillus before NICU discharge. Multiple parameters indicating a prolonged, complicated NICU course were associated with resistant colonization, including gestational age, length of stay, and exposure to several classes of antibiotics. Colonization with resistant bacilli occurred as early as the first NICU day, but acquisition continued throughout the infants' stay. A total of 436 isolates were analyzed by PFGE. On the basis of this molecular analysis, it was determined that duration of colonization was usually very short; the median for all species tested was <1 week. In addition, cross-colonization occurred in only 12% of all PFGE-analyzed isolates. Most clusters of cross-colonized infants were small, with the majority involving only 2 patients.
During endemic periods, acquisition of antibiotic-resistant Gram-negative bacilli in the NICU may occur very soon after admission, but colonization continues over many weeks of NICU stay. The duration of colonization with resistant bacilli is short, and horizontal transmission is unusual. These characteristics suggest a gradual but temporary incorporation of these organisms from the NICU environment into the nascent newborn microflora over time with little cross-colonization. These observations may aid the rational development of infection-control strategies to contain the reservoir of resistant Gram-negative organisms in the NICU.antibiotic resistance, Gram-negative bacilli, neonatal intensive care, antibiotic utilization, colonization, pulsed-field gel electrophoresis.
Dynamic color and brightness adaptation are crucial for visual functioning. The effects of glaucoma on retinal ganglion cells (RGCs) could compromise these functions. We have previously used slow ...dynamic changes of light at moderate intensities to measure the speed and magnitude of subtractive adaptation in RGCs. We used the same procedure to test if RGC abnormalities cause slower and weaker adaptation for patients with glaucoma when compared to age-similar controls. We assessed adaptation deficits in specific classes of RGCs by testing along the three cardinal color axes that isolate konio, parvo, and magno RGCs.
For one eye each of 10 primary open-angle glaucoma patients and their age-similar controls, we measured the speed and magnitude of adapting to 1/32 Hz color modulations along the three cardinal axes, at central fixation and 8° superior, inferior, nasal, and temporal to fixation.
In all 15 comparisons (5 locations × 3 color axes), average adaptation was slower and weaker for glaucoma patients than for controls. Adaptation developed slower at central targets than at 8° eccentricities for controls, but not for patients. Adaptation speed and magnitude differed between affected and control eyes even at retinal locations showing no visual field loss with clinical perimetry.
Neural adaptation is weaker in glaucoma patients for all three classes of RGCs. Since adaptation abnormalities are manifested even at retinal locations not exhibiting a visual field loss, this novel form of assessment may offer a functional insight into glaucoma and an early diagnosis tool.
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