Bodily Sensory Inputs and Anomalous Bodily Experiences in Complex Regional Pain Syndrome: Evaluation of the Potential Effects of Sound Feedback

Neuroscientific studies have shown that human's mental body representations are not fixed but are constantly updated through sensory feedback, including sound feedback. This suggests potential new therapeutic sensory approaches for patients experiencing body-perception disturbances (BPD). BPD can occur in association with chronic pain, for example in Complex Regional Pain Syndrome (CRPS). BPD often impacts on emotional, social, and motor functioning. Here we present the results from a proof-of-principle pilot study investigating the potential value of using sound feedback for altering BPD and its related emotional state and motor behavior in those with CRPS. We build on previous findings that real-time alteration of the sounds produced by walking can alter healthy people's perception of their own body size, while also resulting in more active gait patterns and a more positive emotional state. In the present study we quantified the emotional state, BPD, pain levels and gait of twelve people with CRPS Type 1, who were exposed to real-time alteration of their walking sounds. Results confirm previous reports of the complexity of the BPD linked to CRPS, as participants could be classified into four BPD subgroups according to how they mentally visualize their body. Further, results suggest that sound feedback may affect the perceived size of the CRPS affected limb and the pain experienced, but that the effects may differ according to the type of BPD. Sound feedback affected CRPS descriptors and other bodily feelings and emotions including feelings of emotional dominance, limb detachment, position awareness, attention and negative feelings toward the limb. Gait also varied with sound feedback, affecting the foot contact time with the ground in a way consistent with experienced changes in body weight. Although, findings from this small pilot study should be interpreted with caution, they suggest potential applications for regenerating BDP and its related bodily feelings in a clinical setting for patients with chronic pain and BPD

Prenyl Ammonium Salts – New Carriers for Gene Delivery: A B16-F10 Mouse Melanoma Model

Purpose Prenyl ammonium iodides (Amino-Prenols, APs), semi-synthetic polyprenol derivatives were studied as prospective novel gene transfer agents. Methods AP-7, -8, -11 and -15 (aminoprenols composed of 7, 8, 11 or 15 isoprene units, respectively)were examined for their capacity to form complexes with pDNA, for cytotoxicity and ability to transfect genes to cells. Results All the carriers were able to complex DNA. The highest, comparable to commercial reagents, transfection efficiency was observed for AP-15. Simultaneously, AP-15 exhibited the lowest negative impact on cell viability and proliferation—considerably lower than that of commercial agents. AP-15/DOPE complexes were also efficient to introduce pDNA to cells, without much effect on cell viability. Transfection with AP-15/DOPE complexes influenced the expression of a very few among 44 tested genes involved in cellular lipid metabolism. Furthermore, complexes containing AP-15 and therapeutic plasmid, encoding the TIMP metallopeptidase inhibitor 2 (TIMP2), introduced the TIMP2 gene with high efficiency to B16-F10 melanoma cells but not to B16-F10 melanoma tumors in C57BL/6 mice, as confirmed by TIMP2 protein level determination. Conclusion Obtained results indicate that APs have a potential as non-viral vectors for cell transfection

Cytotoxic Efficacy and Resistance Mechanism of a TRAIL and VEGFA-Peptide Fusion Protein in Colorectal Cancer Models

TNF-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein capable of selectively inducing apoptosis in cancer cells by binding to its cognate receptors. Here, we examined the anticancer efficacy of a recently developed chimeric AD-O51.4 protein, a TRAIL fused to the VEGFA-originating peptide. We tested AD-O51.4 protein activity against human colorectal cancer (CRC) models and investigated the resistance mechanism in the non-responsive CRC models. The quantitative comparison of apoptotic activity between AD-O51.4 and the native TRAIL in nine human colorectal cancer cell lines revealed dose-dependent toxicity in seven of them; the immunofluorescence-captured receptor abundance correlated with the extent of apoptosis. AD-O51.4 reduced the growth of CRC patient-derived xenografts (PDXs) with good efficacy. Cell lines that acquired AD-O51.4 resistance showed a significant decrease in surface TRAIL receptor expression and apoptosis-related proteins, including Caspase-8, HSP60, and p53. These results demonstrate the effectiveness of AD-O51.4 protein in CRC preclinical models and identify the potential mechanism underlying acquired resistance. Progression of AD-O51.4 to clinical trials is expected

Number of viable cells in samples treated with the various APs / AP-based reagents alone (grey bars: AP-7, AP-8, AP-11, AP-15, AP-15/DOPE, AP-15/DOPE/DMEM) and AP:pGFP / AP-15 and DOPE-containing complexes (patterned bars: AP:pGFP: AP-7, AP-8, AP-11 r = 1.7, AP-15 r = 2.0; AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP 2.5 μg of lipids/μg of pDNA), analysed by FACS.

<p>Transfections were performed with the use of 4 μg of pDNA. *P<0.05.</p

Challenges in Stratifying the Molecular Variability of Patient-Derived Colon Tumor Xenografts

Colorectal cancer (CRC) is the second most common cancer in Europe and a leading cause of death worldwide. Patient-derived xenograft (PDX) models maintain complex intratumoral biology and heterogeneity and therefore remain the platform of choice for translational drug discovery. In this study, we implanted 37 primary CRC tumors and five CRC cell lines into NU/J mice to develop xenograft models. Primary tumors and established xenografts were histologically assessed and surveyed for genetic variants and gene expression using a panel of 409 cancer-related genes and RNA-seq, respectively. More than half of CRC tumors (20 out of 37, 54%) developed into a PDX. Histological assessment confirmed that PDX grading, stromal components, inflammation, and budding were consistent with those of the primary tumors. DNA sequencing identified an average of 0.14 variants per gene per sample. The percentage of mutated variants in PDXs increased with successive passages, indicating a decrease in clonal heterogeneity. Gene Ontology analyses of 4180 differentially expressed transcripts (adj. p value < 0.05) revealed overrepresentation of genes involved in cell division and catabolic processes among the transcripts upregulated in PDXs; downregulated transcripts were associated with GO terms related to extracellular matrix organization, immune responses, and angiogenesis. Neither a transcriptome-based consensus molecular subtype (CMS) classifier nor three other predictors reliably matched PDX molecular subtypes with those of the primary tumors. In sum, both genetic and transcriptomic profiles differed between donor tumors and PDXs, likely as a consequence of subclonal evolution at the early phase of xenograft development, making molecular stratification of PDXs challenging

Evaluation of transfection efficiency of B16-F10 cells transfected with use of carrier:pDNA complexes.

<p>(a) Percentage of GFP-positive cells transfected with complexes: AP:pGFP at different N/P ratios (r = 1.7 for AP-7, AP-8, AP-11, r = 2.0 for AP-15) and AP-15/DOPE:pGFP, AP-15/DOPE/DMEM:pGFP lipoplexes containing 2.5 μg of lipids/μg of pGFP, analysed by FACS; Ap- significant difference from AP-15 treatment, At- significant difference from Attractene treatment, L- significant difference from Lipofectamine treatment, P- significant difference from PEI treatment, (b) Activity of β-galactosidase in cells transfected with complexes: AP-11:pLacZ, AP-15:pLacZ, PEI:pLacZ, at r = 2.0–2.5 N/P ratio, studied by β-Gal test; *P<0.05, **P<0.005. Transfections were performed using 4 μg of respective pDNA.</p

Gel retardation analysis with different carrier:pDNA complexes at various N/P ratios (of amino-group of the carriers to the phosphate group of the nucleic acid).

<p>(a) AP-11:pDNA, (b) AP-15:pDNA, (c) AP-7:pDNA, (d) AP-8:pDNA, (e) PEI:pDNA. M- molecular weight size marker 1kb+.</p

Effect of transfection of B16-F10 cells with AP-15/DOPE:pDNA complexes on expression of lipid metabolism-related gene.

<p>Effect of transfection of B16-F10 cells with AP-15/DOPE:pDNA complexes on expression of lipid metabolism-related gene.</p

TIMP2 protein expression in B16-F10 cells assessed by Western blot in.

<p>(a) cell lysates, (b) media from cells. (c) Control Gapdh protein level in cell lysates. Lines: 1- B16-F10 cells, 2- B16-F10 cells transfected with AP-15/DOPE:pGFP, 3- B16-F10 cells transfected with AP-15/DOPE:pTIMP2.</p