Gene therapy for pyoderma gangrenosum: optimal transfection conditions and effect of drugs on gene delivery in the HaCaT cell line using cationic liposomes

Background/Aims: Pyoderma gangrenosum (PG) is a rare ulcerative skin disease, currently treated empirically with immunosuppression. PG is a good target for gene therapy since the skin is easily accessible. This study used the FDA-approved vector Lipofectamine® 2000 to investigate in vitro transfection of skin keratinocytes. The aim was to determine an optimum transfection protocol, including the effect of drugs currently used to treat PG on the efficiency of gene transfer, since gene therapy is unlikely to be used as monotherapy. Methods: Cells of the HaCaT line were transfected with the lacZ reporter gene, and transgene expression was measured after a given time period. Conditions tested were: relative concentrations of DNA and Lipofectamine®, time from transfection to measurement of expression, pH, and exposure to clinically relevant drugs (hydrocortisone, methotrexate, infliximab). Results: The greatest levels of β-galactosidase expression were observed using a DNA:Lipofectamine® ratio of 1:5 (μg/μl) on day 3 after transfection, using culture medium at pH 7, and in the presence of hydrocortisone. Transfection efficiency was reduced by the presence of methotrexate and not significantly affected by infliximab. Conclusion: Gene therapy is a potential future strategy for the management of PG; this study is a step towards the development of a topical gene-based agent

Silsesquioxane polymer as a potential scaffold for laryngeal reconstruction

Cancer, disease and trauma to the larynx and their treatment can lead to permanent loss of structures critical to voice, breathing and swallowing. Engineered partial or total laryngeal replacements would need to match the ambitious specifications of replicating functionality, outer biocompatibility, and permissiveness for an inner mucosal lining. Here we present porous polyhedral oligomeric silsesquioxane-poly(carbonate urea) urethane (POSS-PCUU) as a potential scaffold for engineering laryngeal tissue. Specifically, we employ a precipitation and porogen leaching technique for manufacturing the polymer. The polymer is chemically consistent across all sample types and produces a foam-like scaffold with two distinct topographies and an internal structure composed of nano- and micro-pores. Whilst the highly porous internal structure of the scaffold contributes to the complex tensile behaviour of the polymer, the surface of the scaffold remains largely non-porous. The low number of pores minimise access for cells, although primary fibroblasts and epithelial cells do attach and proliferate on the polymer surface. Our data show that with a change in manufacturing protocol to produce porous polymer surfaces, POSS-PCUU may be a potential candidate for overcoming some of the limitations associated with laryngeal reconstruction and regeneration

Reconstitution Properties of Thymus Stem Cells in Murine Fetal Liver

Injection of day-12 murine fetal liver cells into thymus lobes of Thy-1 congenic adult recipients results in a wave of thymocyte development. The kinetics of repopulation by donor cells reaches a peak after 20–25 days. The frequency of thymic stem cells (TSC) in day-12 fetal liver was estimated, by limit dilution, as 1 in 4x104 cells. Within 8 hr of injection into a thymus lobe, fetal liver TSC commit to T-cell development, losing stem-cell activity. When fetal liver cells are maintained in culture for 7 days, with no exogenous cytokines added, and then injected intra-thymically (I.T.), thymus recolonization is not observed. However, TSC can be maintained in culture for 7 days with IL-1β, IL-3, IL-6, or LIF added, alone or in combination, with steel factor (SLF). Poisson analysis of fetal liver cells cultured with SLF and IL-3 together revealed a precursor frequency of 1 in 1.8x 105 cells. In contrast, the frequency of TSC in adult bone marrow was estimated by limit dilution as 1 in 12,000 cells

Accelerating topical anaesthesia using microneedles

Background/Aims: Topical anaesthetics reduce pain during venous access procedures in children. However, clinical use is hindered by a significant anaesthetic onset time. Restricted diffusion of the topical anaesthetic through the stratum corneum barrier is the principal reason for the delayed onset. Microneedles can painlessly pierce the skin. This study evaluated microneedle pre-treatment of ex vivo human skin as a means to increase the rate of tetracaine permeation, in order to accelerate the onset of anaesthesia. Methods: Franz-type diffusion cells were used to determine permeation of a commercial tetracaine formulation, Ametop gel, through human skin epidermis. Microneedle-assisted permeation was compared to untreated epidermis. Upon completion of permeation studies the epidermal membranes were visually characterised. Results: At 30 minutes 5.43 μg/cm2 of tetracaine had permeated through untreated membrane compared to 12.13 μg/cm2 in microneedle treated membrane. Insertion of a hypodermic needle created a large single channel in the epidermis (approximately 4250μm2) whilst the punctured surface area following microneedle treatments was estimated to be 75,000μm2. Conclusion: Pre-treatment of skin with microneedles significantly enhances the permeation of tetracaine. Microneedles have the potential to more than halve the onset time for anaesthesia when applying Ametop gel

Evaluating the sensitivity, reproducibility and flexibility of a method to test hard shell capsules intended for use in dry powder inhalers

Pharmaceutical tests for hard shell capsules are designed for orally administered capsules. The use of capsules in dry powder inhalers is widespread and increasing and therefore more appropriate tests are required to ensure quality and determine if these capsules are fit for purpose. This study aims to determine the flexibility, reproducibility and sensitivity of a quantitative method that is designed to evaluate the puncture characteristics of different capsule shell formulations under different climatic conditions. A puncture testing method was used to generate force displacement curves for five capsule formulations that were stored and tested at two different temperatures (5 °C and 19 °C). Force-displacement puncture profiles were reproducible for individual capsule shell formulations. The methodology was able to discriminate between capsules produced using different primary materials i.e. gelatin versus hypromellose, as well as more minor changes to capsule formulation i.e. different material grades and excipients. Reduced temperature increased the forces required for capsule puncture however further work is required to confirm its significance. Results indicate the method provides a reproducible and sensitive means of evaluating capsule puncture. Future studies should validate the methodology at different test sites, using different operators and with different capsule shell formulations

Multicentre analysis of incidental findings on low-resolution CT attenuation correction images : an extended study

Objective: To review new incidental findings detected on low-resolution CT attenuation correction (CTAC) images acquired during single-photon emission CT-CT myocardial perfusion imaging as an extension to our initial study. Methods: CTAC images acquired as part of myocardial perfusion imaging performed using single-photon emission CT at four UK nuclear medicine centres were evaluated as part of a multicentre study. New incidental findings that were considered to be clinically significant were evaluated further. Positive-predictive value (PPV) was determined at the time of definitive diagnosis. Results: Out of 3485 patients, 962 (28%) patients had a positive finding on the CTAC image, of which 824 (24%) were new findings. 84 (2.4%) patients had findings that were considered clinically significant at the time of the CTAC report and which had not been previously diagnosed. However, only 10 (0.29%) of these had findings that were confirmed as clinically significant, with the potential to be detrimental to patient outcome, after follow-up and definitive diagnosis. Conclusion: The overall PPV from all centres over the 2-year period was 12%. Each centre achieved what we considered to be low PPVs with no significant difference between the present and initial studies. The additional data from the combined studies show that, statistically, there is no significant difference between the PPVs from any of the centres. We conclude that routine reporting of CTAC images is not beneficial. Advances in knowledge: This study combined with the previous study offers a unique evaluation of new clinically significant incidental findings on low-resolution CT images in an attempt to determine the benefit of reporting the CTAC images