Production and Evaluation of Antibodies and Phage Display-Derived Peptide Ligands for Immunomagnetic Separation of Mycobacterium bovis

This study describes the development and optimization of an immunomagnetic separation (IMS) method to isolate Mycobacterium bovis cells from lymph node tissues. Gamma-irradiated whole M. bovis AF2122/97 cells and ethanol-extracted surface antigens of such cells were used to produce M. bovis-speci?c polyclonal and monoclonal antibodies in rabbits and mice. They were also used to generate M. bovis-speci?c peptide ligands by phage display biopanning. The various antibodies and peptide ligands obtained were used to coat MyOne tosyl-activated Dynabeads (Life Technologies), singly or in combination, and evaluated for IMS. Initially, M. bovis capture from Middlebrook 7H9 broth suspensions (concentration range, 10 to 10⁵ CFU/ml) was evaluated by IMS combined with an M. bovis-speci?c touchdown PCR. IMS-PCR results and, subsequently, IMS-culture results indicated that the beads with greatest immunocapture capability for M. bovis in broth were those coated simultaneously with a monoclonal antibody and a biotinylated 12-mer peptide. These dually coated beads exhibited minimal capture (mean of 0.36% recovery) of 12 other Mycobacterium spp. occasionally encountered in veterinary tuberculosis (TB) diagnostic laboratories. When the optimized IMS method was applied to various M. bovis-spiked lymph node matrices, it demonstrated excellent detection sensitivities (50% limits of detection of 3.16 and 57.7 CFU/ml of lymph node tissue homogenate for IMS-PCR and IMS-culture, respectively). The optimized IMS method therefore has the potential to improve isolation of M. bovis from lymph nodes and hence the diagnosis of bovine tuberculosis.

SUBLINGUAL CAPTOPRIL - A PHARMACOKINETIC AND PHARMACODYNAMIC EVALUATION

In this study we compared the pharmacokinetics and pharmacodynamics of captopril after sublingual and peroral administration. Single 25 mg doses of captopril were administered sublingually and perorally on two different occasions in a randomised cross-over fashion to eight healthy volunteers aged 22-35 years. The kinetics of unchanged captopril, plasma renin activity (PRA), BP and heart rate were studied over three hours after both peroral and sublingual administration of captopril.

Evaluation of c-FLIP in castrate-resistant prostate cancer antagonizes therapeutic response to androgen receptor-targeted therapy

Purpose: To characterize the importance of cellular Fas-associated death domain (FADD)–like interleukin 1ß-converting enzyme (FLICE) inhibitory protein (c-FLIP), a key regulator of caspase-8 (FLICE)–promoted apoptosis, in modulating the response of prostate cancer cells to androgen receptor (AR)–targeted therapy.

Experimental Design: c-FLIP expression was characterized by immunohistochemical analysis of prostatectomy tissue. The functional importance of c-FLIP to survival and modulating response to bicalutamide was studied by molecular and pharmacologic interventions.

Results: c-FLIP expression was increased in high-grade prostatic intraepithelial neoplasia and prostate cancer tissue relative to normal prostate epithelium (P < 0.001). Maximal c-FLIP expression was detected in castrate-resistant prostate cancer (CRPC; P < 0.001). In vitro, silencing of c-FLIP induced spontaneous apoptosis and increased 22Rv1 and LNCaP cell sensitivity to bicalutamide, determined by flow cytometry, PARP cleavage, and caspase activity assays. The histone deacetylase inhibitors (HDACi), droxinostat and SAHA, also downregulated c-FLIP expression, induced caspase-8- and caspase-3/7–mediated apoptosis, and increased apoptosis in bicalutamide-treated cells. Conversely, the elevated expression of c-FLIP detected in the CRPC cell line VCaP underpinned their insensitivity to bicalutamide and SAHA in vitro. However, knockdown of c-FLIP induced spontaneous apoptosis in VCaP cells, indicating its relevance to cell survival and therapeutic resistance.

Conclusion: c-FLIP reduces the efficacy of AR-targeted therapy and maintains the viability of prostate cancer cells. A combination of HDACi with androgen deprivation therapy may be effective in early-stage disease, using c-FLIP expression as a predictive biomarker of sensitivity. Direct targeting of c-FLIP, however, may be relevant to enhance the response of existing and novel therapeutics in CRPC. Clin Cancer Res; 18(14); 3822–33.

A review and psychometric evaluation of pregnancy-specific stress measures

Considerable evidence has accumulated on the association between pregnancy-specific stress and adverse birth outcomes with an increasing number of measures of pregnancy-specific stress being developed internationally. However, the introduction of these measures has not always been theoretically or psychometrically grounded, resulting in questions about the quality and direction of such research. This review summarizes evidence on the reliability and validity of pregnancy-specific stress measures identified between 1980 and October 2010. Fifteen pregnancy-specific stress measures were identified. Cronbach’s alpha coefficient ranged from 0.51–0.96 and predictive validity data on preterm birth were reported for five measures. Convergent validity data suggest that pregnancy-specific stress is related to, but distinct from, global stress. Findings from this review consolidate current knowledge on pregnancy-specific stress as a consistent predictor of premature birth. This review also advances awareness of the range of measures of pregnancy-specific stress and documents their strengths and limitations based on published reliability and validity data. Careful consideration needs to be given as to which measures to use in future research to maximize the development of stress theory in pregnancy and appropriate interventions for women who experience stress in pregnancy. An international, strategic collaboration is recommended to advance knowledge in this area of study.

Construction and evaluation of plasmid vectors optimized for constitutive and regulated gene expression in Burkholderia cepacia complex isolates

Genetic studies with Burkholderia cepacia complex isolates are hampered by the limited availability of cloning vectors and by the inherent resistance of these isolates to the most common antibiotics used for genetic selection. Also, some of the promoters widely employed for gene expression in Escherichia coli are inefficient in B. cepacia. In this study, we have utilized the backbone of the vector pME6000, a derivative of the pBBR1 plasmid that was originally isolated from Bordetella bronchiseptica, to construct a set of vectors useful for gene expression in B. cepacia. These vectors contain either the constitutive promoter of the S7 ribosomal protein gene from Burkholderia sp. strain LB400 or the arabinose-inducible P(BAD) promoter from E. coli. Promoter sequences were placed immediately upstream of multiple cloning sites in combination with the minimal sequence of pME6000 required for plasmid maintenance and mobilization. The functionality of both vectors was assessed by cloning the enhanced green fluorescent protein gene (e-gfp) and determining the levels of enhanced green fluorescent protein expression and fluorescence emission for a variety of clinical and environmental isolates of the B. cepacia complex. We also demonstrate that B. cepacia carrying these constructs can readily be detected intracellularly by fluorescence microscopy following the infection of Acanthamoeba polyphaga.

Novel macrocyclic and acyclic cationic lipids for gene transfer: Synthesis and in vitro evaluation

The synthesis and in vitro evaluation of four cationic lipid gene delivery vectors, characterized by acyclic or macrocyclic, and saturated or unsaturated hydrophobic regions, is described. The synthesis employed standard protocols, including ring-closing metathesis for macrocyclic lipid construction. All lipoplexes studied, formulated from plasmid DNA and a liposome composed of a synthesized lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol as co-lipid, exhibited plasmid DNA binding and protection from DNase I degradation, and concentration dependent cytotoxicity using Chinese hamster ovary-K1 cells. The transfection efficiency of formulations with cholesterol outperformed those with DOPE, and in many cases the EPC/cholesterol control, and formulations with a macrocyclic lipid (+/- 10:1) outperformed their acyclic counterparts (+/- 3:1).

Rapid serological diagnosis of Helicobacter pylori:a need for caution and re-evaluation

Screening for Helicobacter pylori in dyspeptic patients may improve selectivity for gastroscopy. Rapid serological tests based on ELISA technique are cheap, readily available and simple to use in the clinical setting. However local evaluation is essential in order to validate these techniques. Fifty-six dyspeptic patients (aged less than 45 yr) had a rapid serological test (Helisal) performed prior to gastroscopy. At gastroscopy H. pylori status was assessed using culture and histology. The Helisal sensitivity was 80 per cent, specificity 82 per cent. Screening patients with the Helisal test would have missed 6 patients with peptic ulcer disease and 2 with oesophagitis. The Helisal test did not perform satisfactorily as a screening test in selection of patients for gastroscopy.

The Evaluation of an Interdisciplinary Pain Protocol in Long Term Care

Objectives: To evaluate the effectiveness of (1) dissemination strategies to improve clinical practice behaviors (eg, frequency and documentation of pain assessments, use of pain medication) among health care team members, and (2) the implementation of the pain protocol in reducing pain in long term care (LTC) residents. Design: A controlled before-after design was used to evaluate the effectiveness of the pain protocol, whereas qualitative interviews and focus groups were used to obtain additional context-driven data. Setting: Four LTC facilities in southern Ontario, Canada; 2 for the intervention group and 2 for the control group. Participants: Data were collected from 200 LTC residents; 99 for the intervention and 101 for the control group. Intervention: Implementation of a pain protocol using a multifaceted approach, including a site working group or Pain Team, pain education and skills training, and other quality improvement activities. Measurements: Resident pain was measured using 3 assessment tools: the Pain Assessment Checklist for Seniors with Limited Ability to Communicate, the Pain Assessment in the Communicatively Impaired Elderly, and the Present Pain Intensity Scale. Clinical practice behaviors were measured using a number of process indicators; for example, use of pain assessment tools, documentation about pain management, and use of pain medications. A semistructured interview guide was used to collect qualitative data via focus groups and interviews. Results: Pain increased significantly more for the control group than the intervention group over the 1-year intervention period. There were significantly more positive changes over the intervention period in the intervention group compared with the control group for the following indicators: the use of a standardized pain assessment tool and completed admission/initial pain assessment. Qualitative findings highlight the importance of reminding staff to think about pain as a priority in caring for residents and to be mindful of it during daily activities. Using onsite champions, in this case advanced practice nurses and a Pain Team, were key to successfully implementing the pain protocol. Conclusions: These study findings indicate that the implementation of a pain protocol intervention improved the way pain was managed and provided pain relief for LTC residents.

Evaluation of quality of life and priorities of patients with glaucoma

PURPOSE: To investigate the quality of life and priorities of patients with glaucoma.

METHODS: Patients diagnosed with glaucoma and no other ocular comorbidity were consecutively recruited. Clinical information was collected. Participants were asked to complete three questionnaires: EuroQuol (EQ-5D), time tradeoff (TTO), and choice-based conjoint analysis. The latter used five-attribute outcomes: (1) reading and seeing detail, (2) peripheral vision, (3) darkness and glare, (4) household chores, and (5) outdoor mobility. Visual field loss was estimated by using binocular integrated visual fields (IVFs).

RESULTS: Of 84 patients invited to participate, 72 were enrolled in the study. The conjoint utilities showed that the two main priorities were "reading and seeing detail" and "outdoor mobility." This rank order was stable across all segmentations of the data by demographic or visual state. However, the relative emphasis of these priorities changed with increasing visual field loss, with concerns for central vision increasing, whereas those for outdoor mobility decreased. Two subgroups of patients with differing priorities on the two main attributes were identified. Only 17% of patients (those with poorer visual acuity) were prepared to consider TTO. A principal component analysis revealed relatively independent components (i.e., low correlations) between the three different methodologies for assessing quality of life.

CONCLUSIONS: Assessments of quality of life using different methodologies have been shown to produce different outcomes with low intercorrelations between them. Only a minority of patients were prepared to trade time for a return to normal vision. Conjoint analysis showed two subgroups with different priorities. Severity of glaucoma influenced the relative importance of priorities.

Surveillance for ocular hypertension: An evidence synthesis and economic evaluation

OBJECTIVES: To determine effective and efficient monitoring criteria for ocular hypertension [raised intraocular pressure (IOP)] through (i) identification and validation of glaucoma risk prediction models; and (ii) development of models to determine optimal surveillance pathways.

DESIGN: A discrete event simulation economic modelling evaluation. Data from systematic reviews of risk prediction models and agreement between tonometers, secondary analyses of existing datasets (to validate identified risk models and determine optimal monitoring criteria) and public preferences were used to structure and populate the economic model.

SETTING: Primary and secondary care.

PARTICIPANTS: Adults with ocular hypertension (IOP > 21 mmHg) and the public (surveillance preferences).

INTERVENTIONS: We compared five pathways: two based on National Institute for Health and Clinical Excellence (NICE) guidelines with monitoring interval and treatment depending on initial risk stratification, 'NICE intensive' (4-monthly to annual monitoring) and 'NICE conservative' (6-monthly to biennial monitoring); two pathways, differing in location (hospital and community), with monitoring biennially and treatment initiated for a ≥ 6% 5-year glaucoma risk; and a 'treat all' pathway involving treatment with a prostaglandin analogue if IOP > 21 mmHg and IOP measured annually in the community.

MAIN OUTCOME MEASURES: Glaucoma cases detected; tonometer agreement; public preferences; costs; willingness to pay and quality-adjusted life-years (QALYs).

RESULTS: The best available glaucoma risk prediction model estimated the 5-year risk based on age and ocular predictors (IOP, central corneal thickness, optic nerve damage and index of visual field status). Taking the average of two IOP readings, by tonometry, true change was detected at two years. Sizeable measurement variability was noted between tonometers. There was a general public preference for monitoring; good communication and understanding of the process predicted service value. 'Treat all' was the least costly and 'NICE intensive' the most costly pathway. Biennial monitoring reduced the number of cases of glaucoma conversion compared with a 'treat all' pathway and provided more QALYs, but the incremental cost-effectiveness ratio (ICER) was considerably more than £30,000. The 'NICE intensive' pathway also avoided glaucoma conversion, but NICE-based pathways were either dominated (more costly and less effective) by biennial hospital monitoring or had a ICERs > £30,000. Results were not sensitive to the risk threshold for initiating surveillance but were sensitive to the risk threshold for initiating treatment, NHS costs and treatment adherence.

LIMITATIONS: Optimal monitoring intervals were based on IOP data. There were insufficient data to determine the optimal frequency of measurement of the visual field or optic nerve head for identification of glaucoma. The economic modelling took a 20-year time horizon which may be insufficient to capture long-term benefits. Sensitivity analyses may not fully capture the uncertainty surrounding parameter estimates.

CONCLUSIONS: For confirmed ocular hypertension, findings suggest that there is no clear benefit from intensive monitoring. Consideration of the patient experience is important. A cohort study is recommended to provide data to refine the glaucoma risk prediction model, determine the optimum type and frequency of serial glaucoma tests and estimate costs and patient preferences for monitoring and treatment.

FUNDING: The National Institute for Health Research Health Technology Assessment Programme.

Evaluation of corneal endothelium and keratic precipitates by specular microscopy in anterior uveitis

Background - The study of corneal endothelium, by specular microscopy, in patients with anterior uveitis has largely been restricted to observations on the endothelial cells. In this prospective study 'keratic precipitates' (KP) in different types of uveitis were examined in different stages of the disease process and the endothelial changes occurring in the vicinity of the KP were evaluated in comparison with the endothelium of the uninvolved eye. Methods - 13 patients with active unilateral uveitis were recruited. The mean age was 42.9 years (range 20-76 years). A Tomey-1100 contact wide field specular (x10) microscope was used to capture endothelial images and KP until the resolution of uveitis. Data regarding type of uveitis, number, size, and nature of KP were recorded. Automated morphometric analysis was done for cell size, cell density and coefficient of variation, and statistical comparisons of cell size and cell density were made (Student's t test) between the endothelium in the vicinity of fresh and resolving KP, fresh KP and normal endothelium, and resolving KP and normal endothelium. Results - On specular microscopy, fresh KP were seen as dense, white glistening deposits occupying 5-10 endothelial cells in diameter and fine KP were widely distributed and were one or two endothelial cells in diameter. The KP in Posner-Schlossman syndrome had a distinct and different morphology. With clinical remission of uveitis, the KP were observed to undergo characteristic morphological changes and old KP demonstrated a large, dark halo surrounding a central white deposit and occasionally a dark shadow or a 'lacuna' replaced the site of the original KP. Endothelial blebs were noted as dark shadows or defects in the endothelial mosaic in patients with recurrent uveitis. There was significant statistical difference in the mean cell size and cell density of endothelial cells in the vicinity of fresh KP compared with normal endothelium of the opposite eye. Conclusion - This study elucidated the different specular microscopic features of KP in anterior uveitis. Distinct morphological features of large and fine KP were noted. These features underwent dramatic changes on resolution of uveitis. The endothelium was abnormal in the vicinity of KP, which returned to near normal values on resolution of uveitis.

SMART Arm with outcome-triggered electrical stimulation:a pilot randomized clinical trial

The SMART (SensoriMotor Active Rehabilitation Training) Arm is a nonrobotic device designed to allow stroke survivors with severe paresis to practice reaching. It can be used with or without outcome-triggered electrical stimulation (OT-stim) to augment movement. The aim of this study was to evaluate the efficacy of SMART Arm training when used with or without OT-stim, in addition to usual care, as compared with usual care alone during inpatient rehabilitation.

Evaluation of Microcollimated Pars Plana External Beam Radiation in the Porcine Eye

Purpose: To evaluate the clinical and histological side effects of a prototype stereotactic radiotherapy system delivering microcollimated external beam radiation through pars plana in porcine eyes.

Methods: Five Yucatan mini-swine (10 eyes) were randomized to five treatment groups. Eight eyes were dosed with X-ray radiation on Day 1, and two eyes served as untreated controls. Treated eyes received doses up to 60 Gy to the retina and up to 130 Gy to the sclera using single or overlapping beams. The treatment beams were highly collimated such that the diameter was approximately 2.5 mm on the sclera and 3 mm on the retinal surface. Fundus photography, fluorescein angiography (FA), and spectral domain optical coherence tomography (SD-OCT) were obtained on days 7, 30, 60, and 110. Images were examined by a masked grader and evaluated for abnormalities. Animals were sacrificed on day 111 and gross and histopathological analysis was conducted.

Results: Histological and gross changes to eye structures including conjunctiva and lens were minimal at all doses. Fundus, FA, and SD-OCT of the targeted region failed to disclose any abnormality in the control or 21 Gy treated animals. In the 42 and 60 Gy animals, hypopigmented spots were noted after treatment on clinical exam, and corresponding hyperfluorescent staining was seen in late frames. No evidence of choroidal hypoperfusion was seen. The histological specimens from the 60 Gy animals showed photoreceptor loss and displacement of cone nuclei.

Conclusion: Transcleral stereotactic radiation dosing in porcine eyes can be accomplished with no significant adverse events as doses less than 42 Gy.