Parents' perception of self-advocacy of children with myositis: an anonymous online survey

<p>Abstract</p> <p>Background</p> <p>Children with complex medical issues experience barriers to the transition of care from pediatric to adult providers. We sought to identify these barriers by elucidating the experiences of patients with idiopathic inflammatory muscle disorders.</p> <p>Methods</p> <p>We collected anonymous survey data using an online website. Patients and their families were solicited from the US and Canada through established clinics for children with idiopathic inflammatory muscle diseases as well as with the aid of a nonprofit organization for the benefit of such individuals. The parents of 45 older children/young adults suffering from idiopathic inflammatory muscle diseases were surveyed. As a basis of comparison, we similarly collected data from the parents of 207 younger children with inflammatory muscle diseases. The survey assessed transition of care issues confronting families of children and young adults with chronic juvenile myositis.</p> <p>Results</p> <p>Regardless of age of the patient, respondents were unlikely to have a designated health care provider assigned to aid in transition of care and were unlikely to be aware of a posted policy concerning transition of care at their pediatrician's office. Additionally, regardless of age, patients and their families were unlikely to have a written plan for moving to adult care.</p> <p>Conclusions</p> <p>We identified deficiencies in the health care experiences of families as pertain to knowledge, self-advocacy, policy, and vocational readiness. Moreover, as children with complex medical issues grow up, parents attribute less self-advocacy to their children's level of independence.</p

Distinct effects of endosomal escape and inhibition of endosomal trafficking on gene delivery via electrotransfection.

A recent theory suggests that endocytosis is involved in uptake and intracellular transport of electrotransfected plasmid DNA (pDNA). The goal of the current study was to understand if approaches used previously to improve endocytosis of gene delivery vectors could be applied to enhancing electrotransfection efficiency (eTE). Results from the study showed that photochemically induced endosomal escape, which could increase poly-L-lysine (PLL)-mediated gene delivery, decreased eTE. The decrease could not be blocked by treatment of cells with endonuclease inhibitors (aurintricarboxylic acid and zinc ion) or antioxidants (L-glutamine and ascorbic acid). Chemical treatment of cells with an endosomal trafficking inhibitor that blocks endosome progression, bafilomycin A1, resulted in a significant decrease in eTE. However, treatment of cells with lysosomotropic agents (chloroquine and ammonium chloride) had little effects on eTE. These data suggested that endosomes played important roles in protecting and intracellular trafficking of electrotransfected pDNA

Enhancing Electrotransfection Efficiency through Improvement in Nuclear Entry of Plasmid DNA

The nuclear envelope is a physiological barrier to electrogene transfer. To understand different mechanisms of the nuclear entry for electrotransfected plasmid DNA (pDNA), the current study investigated how manipulation of the mechanisms could affect electrotransfection efficiency (eTE), transgene expression level (EL), and cell viability. In the investigation, cells were first synchronized at G2-M phase prior to electrotransfection so that the nuclear envelope breakdown (NEBD) occurred before pDNA entered the cells. The NEBD significantly increased the eTE and the EL while the cell viability was not compromised. In the second experiment, the cells were treated with a nuclear pore dilating agent (i.e., trans-1,2-cyclohexanediol). The treatment could increase the EL, but had only minor effects on eTE. Furthermore, the treatment was more cytotoxic, compared with the cell synchronization. In the third experiment, a nuclear targeting sequence (i.e., SV40) was incorporated into the pDNA prior to electrotransfection. The incorporation was more effective than the cell synchronization for enhancing the EL, but not the eTE, and the effectiveness was cell type dependent. Taken together, the data described above suggested that synchronization of the NEBD could be a practical approach to improving electrogene transfer in all dividing cells

Effects of lysosomotropic agents pretreatment on transgene expression.

<p> COS7 and HEK293 cells were treated with either buffer in the control (Ctrl) or a lysosomotropic agent for 4 hours prior to electrotransfection: (a) chloroquine (CQ) at 100 μM, and (b) ammonium chloride (AC) at 10 mM. The treatments had statistically insignificant effects on eTE quantified at 24 hours post electrotransfection. (n = 4, P > 0.05, Mann-Whitney U test).</p

Efficiency of cell transfection with Poly-L-lysine (PLL).

<p>Intracellular vesicles were preloaded with the photosensitizer. COS7 cells were transfected with PLL-pDNA complexes for 4 hours at 37°C. Then, the cells were exposed to blue light for 0 (i.e., no light (NL) control), 1 min, and 5 min. The transfection efficiency (TE) was quantified at 24 hours post transfection. (n = 4, *P<0.05, Mann-Whitney U test).</p

Effects of bafilomycin A1 pretreatment on transgene expression.

<p> COS7 and HEK293 cells were treated with either buffer in the control (Ctrl) or an endocytic trafficking inhibitor, bafilomycin A1 (Baf A1), at 1 μM for 1 hour prior to electrotransfection; and the eTE was quantified at 24 hours post electrotransfection. (n = 4, *P<0.05, Mann-Whitney U test).</p

Release of rhodamine B-labeled dextran from endosomes following light treatment.

<p>Intracellular vesicles were preloaded with the photosensitizer. (a) The image of a COS7 cell before light treatment. It was taken at 20 min after the cell was incubated at 37°C with dextran (10,000 MW). The light treatment was performed immediately after the image acquisition. (b) The image of the same cell at 10 min after light treatment. The dextran was punctate within endosomes before light treatment (top panel). However, upon light treatment, dextran diffused out of endosomes, and spread in the cytosol (bottom panel).</p