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Sampson J.H.,Duke University | Archer G.,Duke University | Pedain C.,BrainLAB | Wembacher-Schroder E.,BrainLAB | And 73 more authors.
Journal of Neurosurgery | Year: 2010

Object. Convection-enhanced delivery (CED) is a novel intracerebral drug delivery technique with considerable promise for delivering therapeutic agents throughout the CNS. Despite this promise, Phase III clinical trials employing CED have failed to meet clinical end points. Although this may be due to inactive agents or a failure to rigorously validate drug targets, the authors have previously demonstrated that catheter positioning plays a major role in drug distribution using this technique. The purpose of the present work was to retrospectively analyze the expected drug distribution based on catheter positioning data available from the CED arm of the PRECISE trial. Methods. Data on catheter positioning from all patients randomized to the CED arm of the PRECISE trial were available for analyses. BrainLAB iPlan Flow software was used to estimate the expected drug distribution. Results. Only 49.8% of catheters met all positioning criteria. Still, catheter positioning score (hazard ratio 0.93, p = 0.043) and the number of optimally positioned catheters (hazard ratio 0.72, p = 0.038) had a significant effect on progression-free survival. Estimated coverage of relevant target volumes was low, however, with only 20.1% of the 2-cm penumbra surrounding the resection cavity covered on average. Although tumor location and resection cavity volume had no effect on coverage volume, estimations of drug delivery to relevant target volumes did correlate well with catheter score (p >0.003), and optimally positioned catheters had larger coverage volumes (p < 0.002). Only overall survival (p = 0.006) was higher for investigators considered experienced after adjusting for patient age and Karnofsky Performance Scale score. Conclusions. The potential efficacy of drugs delivered by CED may be severely constrained by ineffective delivery in many patients. Routine use of software algorithms and alternative catheter designs and infusion parameters may improve the efficacy of drugs delivered by CED.

Hamula C.L.A.,University of Alberta | Hamula C.L.A.,Mount Sinai School of Medicine | Peng H.,University of Alberta | Wang Z.,University of Alberta | And 4 more authors.
Methods | Year: 2015

Streptococcus pyogenes is a clinically important pathogen consisting of various serotypes determined by different M proteins expressed on the cell surface. The M type is therefore a useful marker to monitor the spread of invasive S. pyogenes in a population. Serotyping and nucleic acid amplification/sequencing methods for the identification of M types are laborious, inconsistent, and usually confined to reference laboratories. The primary objective of this work is to develop a technique that enables generation of aptamers binding to specific M-types of S. pyogenes. We describe here an in vitro technique that directly used live bacterial cells and the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) strategy. Live S. pyogenes cells were incubated with DNA libraries consisting of 40-nucleotides randomized sequences. Those sequences that bound to the cells were separated, amplified using polymerase chain reaction (PCR), purified using gel electrophoresis, and served as the input DNA pool for the next round of SELEX selection. A specially designed forward primer containing extended polyA20/5Sp9 facilitated gel electrophoresis purification of ssDNA after PCR amplification. A counter-selection step using non-target cells was introduced to improve selectivity. DNA libraries of different starting sequence diversity (1016 and 1014) were compared. Aptamer pools from each round of selection were tested for their binding to the target and non-target cells using flow cytometry. Selected aptamer pools were then cloned and sequenced. Individual aptamer sequences were screened on the basis of their binding to the 10 M-types that were used as targets. Aptamer pools obtained from SELEX rounds 5-8 showed high affinity to the target S. pyogenes cells. Tests against non-target Streptococcus bovis, Streptococcus pneumoniae, and Enterococcus species demonstrated selectivity of these aptamers for binding to S. pyogenes. Several aptamer sequences were found to bind preferentially to the M11 M-type of S. pyogenes. Estimated binding dissociation constants (Kd ) were in the low nanomolar range for the M11 specific sequences; for example, sequence E-CA20 had a Kd of 7±1nM. These affinities are comparable to those of a monoclonal antibody. The improved bacterial cell-SELEX technique is successful in generating aptamers selective for S. pyogenes and some of its M-types. These aptamers are potentially useful for detecting S. pyogenes, achieving binding profiles of the various M-types, and developing new M-typing technologies for non-specialized laboratories or point-of-care testing. © 2015 Elsevier Inc.

Gounder P.P.,Centers for Disease Control and Prevention | Zulz T.,Centers for Disease Control and Prevention | Desai S.,Public Health Agency of Canada | Stenz F.,The National Board of Health | And 4 more authors.
Journal of Infection | Year: 2015

Objective: To determine the incidence of meningitis caused by Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae in the North American Arctic during 2000-2010. Methods: Surveillance data were obtained from the International Circumpolar Surveillance network. We defined a case of bacterial meningitis caused by H. influenzae, N. meningitidis, or S. pneumoniae as a culture-positive isolate obtained from a normally sterile site in a resident with a meningitis diagnosis. Results: The annual incidence/100,000 persons for meningitis caused by H. influenzae, N. meningitidis, and S. pneumoniae among all North American Arctic residents was: 0.6, 0.5, and 1.5, respectively; the meningitis incidence among indigenous persons in Alaska and Canada (indigenous status not recorded in Greenland) for those three bacteria was: 2.1, 0.8, and 2.4, respectively. The percentage of pneumococcal isolates belonging to a 7-valent pneumococcal conjugate vaccine serotype declined from 2000-2004 to 2005-2010 (31%-2%, p-value <0.01). During 2005-2010, serotype a caused 55% of H. influenzae meningitis and serogroup B caused 86% of meningococcal meningitis. Conclusions: Compared with all North American Arctic residents, indigenous people suffer disproportionately from bacterial meningitis. Arctic residents could benefit from the development of an H. influenzae serotype a vaccine and implementation of a meningococcal serogroup B vaccine. © 2015.

Hamula C.L.A.,University of Alberta | Hamula C.L.A.,Mount Sinai School of Medicine | Peng H.,University of Alberta | Wang Z.,University of Alberta | And 5 more authors.
Journal of Molecular Evolution | Year: 2015

Aptamers of high affinity and specificity have a wide range of analytic and clinical applications. Selection of DNA or RNA aptamer molecules usually involves systematic evolution of ligands via exponential enrichment (SELEX), in which a random DNA or RNA library is incubated with a target molecule, and the oligonucleotides that bind the target are then separated from the nonbinders, PCR amplified, and used as refined libraries in the next round of selection. Conventional SELEX methodologies require the use of purified target molecules and their immobilization onto a solid support. However, purified targets from cells are not always available, and fixing the target to a support may alter its conformation. To overcome these problems, we have developed a SELEX technique using live bacterial cells in suspension as targets, for selecting DNA aptamers specific to cell-surface molecules. Through the selection of aptamers binding to Lactobacillus acidophilus and Streptococcus pyogenes, we report here optimization of this technique and show how varying selection conditions impact the characteristics of resultant aptamer pools, including the binding affinity, selectivity, and the secondary structures. We found that the use of larger starting library sequence diversity, gel purification of the subsequent pools, and the introduction of counter-selection resulted in a more efficient SELEX process and more selective aptamers. A SELEX protocol with lower starting sequence diversity, the use of heat denaturation, and the absence of counter-selection still resulted in high-affinity aptamer sequences specific to the target cell types; however, the SELEX process was inefficient, requiring 20 rounds, and the aptamers were not specific to the strain of the bacterial cells. Strikingly, two different SELEX methodologies yielded the same sequence that bound strongly to the target S. pyogenes cells, suggesting the robustness of the bacterial cell-SELEX technique. © 2015, Springer Science+Business Media New York.

Lu C.,University of Alberta | Mahmood M.,Walter Mackenzie Health science Center | Jha N.,University of Alberta | Mandal M.,University of Alberta
Pattern Recognition | Year: 2013

In the diagnosis of skin melanoma by analyzing histopathological images, the detection of the melanocytes in the epidermis area is an important step. However, the detection of the melanocytes from the epidermis area is difficult because other keratinocytes that are very similar to the melanocytes are also present. This paper proposes a novel computer-aided technique for detection of the melanocytes in the epidermis area of skin histopathological images. An adaptive threshold technique is first applied to segment all the keratinocytes in the image. In order to distinguish the melanocytes from other keratinocytes, a novel technique based on radial line scanning is proposed to estimate the halo region of the melanocytes. Based on the estimated halo region of all the nuclei, an area ratio of estimated halo region and the nuclei is used to detect the melanocytes from all the keratinocytes. Experimental results on 40 different histopathological images of skin tissue containing 341 melanocytes show that the proposed technique provides a superior performance. © 2012 Elsevier Ltd.

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