K. E. Wright

Experimental therapies for repair of the central nervous system: stem cells and tissue engineering.

Several stem cell‐based therapeutic tools are currently being investigated for the regeneration of central nervous system (CNS) injuries. This review focuses on innovative approaches for CNS tissue repair via the use of implantable cellular devices. These devices are supported by biopharmaceuticals and conventional physiotherapy for the restoration of lost neuronal circuits and CNS function. This paper further reviews new and promising tools currently in pre‐clinical and clinical tests for the treatment of CNS diseases where substantial loss of cellular and extracellular components of neural tissue has occurred such as stroke, encephalopathy and traumatic neural injuries. We also discuss selected 3D bioscaffolds co‐cultured with clinically applicable human mesenchymal stem cells. Recent advances in neural tissue engineering and stem cell differentiation methods have shown promise for their clinical application in treating yet incurable CNS deficits. Copyright

Peripheral neural cell sensitivity to mTHPC-mediated photodynamic therapy in a 3D in vitro model

Background:The effect of photodynamic therapy (PDT) on neural cells is important when tumours are within or adjacent to the nervous system. The purpose of this study was to investigate PDT using the photosensitiser, meta-tetrahydroxyphenyl chlorin (mTHPC), on rat neurons and satellite glia, compared with human adenocarcinoma cells (MCF-7).Methods:Fluorescence microscopy confirmed that mTHPC was incorporated into all three cell types. Sensitivity of cells exposed to mTHPC-PDT (0–10 μg ml–1) was determined in a novel 3-dimensional collagen gel culture system. Cell death was quantified using propidium iodide and cell types were distinguished using immunocytochemistry. In some cases, neuron survival was confirmed by measuring subsequent neurite growth in monolayer culture.Results:MCF-7s and satellite glia were significantly more sensitive to PDT than neurons. Importantly, 4 μg ml–1 mTHPC-PDT caused no significant neuron death compared with untreated controls but was sufficient to elicit substantial cell death in the other cell types. Initially, treatment reduced neurite length; neurons then extended neurites equivalent to those of untreated controls. The protocol was validated using hypericin (0–3 μg ml–1), which caused neuron death equivalent to other cell types.Conclusion:Neurons in culture can survive mTHPC-PDT under conditions sufficient to kill tumour cells and other nervous system cells.

Encapsulation of Mesenchymal Stem Cells by Bioscaffolds Protects Cell Survival and Attenuates Neuroinflammatory Reaction in Injured Brain Tissue after Transplantation

Since the brain is naturally inefficient in regenerating functional tissue after injury or disease, novel restorative strategies including stem cell transplantation and tissue engineering have to be considered. We have investigated the use of such strategies in order to achieve better functional repair outcomes. One of the fundamental challenges of successful transplantation is the delivery of cells to the injured site while maintaining cell viability. Classical cell delivery methods of intravenous or intraparenchymal injections are plagued by low engraftment and poor survival of transplanted stem cells. Novel implantable devices such as 3D bioactive scaffolds can provide the physical and metabolic support required for successful progenitor cell engraftment, proliferation, and maturation. In this study, we performed in situ analysis of laminin-linked dextran and gelatin macroporous scaffolds. We revealed the protective action of gelatin–laminin (GL) scaffolds seeded with mesenchymal stem cells derived from donated human Whartons jelly (hUCMSCs) against neuroinflammatory reactions of injured mammalian brain tissue. These bioscaffolds have been implanted into (i) intact and (ii) ischemic rat hippocampal organotypic slices and into the striatum of (iii) normal and (iv) focally injured brains of adult Wistar rats. We found that transplantation of hUCMSCs encapsulated in GL scaffolds had a significant impact on the prevention of glial scar formation (low glial acidic fibrillary protein) and in the reduction of neuroinflammation (low interleukin-6 and the microglial markers ED1 and Iba1) in the recipient tissue. Moreover, implantation of hUCMSCs encapsulated within GL scaffolds induced matrix metalloproteinase-2 and -9 proteolytic activities in the surrounding brain tissue. This facilitated scaffold biodegradation while leaving the remaining grafted hUCMSCs untouched. In conclusion, transplanting GL scaffolds preseeded with hUCMSCs into mammalian brain tissue escaped the hosts immune system and protected neural tissue from neuroinflammatory injury. This manuscript is published as part of the International Association of Neurorestoratology (IANR) supplement issue of Cell Transplantation.

Preparation and validation of the first WHO international genetic reference panel for Fragile X syndrome

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by expansion of a trinucleotide (CGG)n repeat sequence in the 5′ untranslated region of the FMR1 gene, resulting in promoter hypermethylation and suppression of FMR1 transcription. Additionally, pre-mutation alleles in carrier males and females may result in Fragile X tremor ataxia syndrome and primary ovarian insufficiency, respectively. Fragile X is one of the most commonly requested molecular genetic tests worldwide. Quality assessment schemes have identified a wide disparity in allele sizing between laboratories. It is therefore important that clinical laboratories have access to characterized reference materials (RMs) to aid accurate allele sizing and diagnosis. With this in mind, a panel of genotyping RMs for Fragile X syndrome has been developed, which should be stable over many years and available to all diagnostic laboratories. Immortalized cell lines were produced by Epstein–Barr virus transformation of lymphocytes from consenting patients. Genomic DNA was extracted in bulk and RM aliquots were freeze-dried in glass ampoules. Twenty-one laboratories from seventeen countries participated in a collaborative study to assess their suitability. Participants evaluated the samples (blinded, in triplicate) in their routine methods alongside in-house and commercial controls. The panel of five genomic DNA samples was endorsed by the European Society of Human Genetics and approved as an International Standard by the Expert Committee on Biological Standardization at the World Health Organization.

Inhibition of specific cellular antioxidant pathways increases the sensitivity of neurons to meta-tetrahydroxyphenyl chlorin-mediated photodynamic therapy in a 3D co-culture model.

The effect of photodynamic therapy (PDT) on neurons is of critical importance when treating cancers within or adjacent to the nervous system. Neurons show reduced sensitivity to meta‐tetrahydroxyphenyl chlorin (mTHPC) mediated PDT, so the aim of this study was to investigate whether neuron sparing is due to endogenous cellular antioxidant activity. Dorsal root ganglion (DRG) neurons and their associated satellite glia were subjected to mTHPC‐PDT in a 3D co‐culture system following incubation with antioxidant inhibitors: diethyl dithiocarbamate (DDC, SOD‐1 inhibitor), 2‐methoxyestradiol (2‐MeOH2, SOD‐2 inhibitor) and l‐buthionine sulfoximine (l‐BSO, glutathione synthase inhibitor). Sensitivity of each cell type was assessed using a combination of live/dead staining and immunofluorescence. Pretreatment with DDC and with l‐BSO significantly increased the sensitivity of neurons to mTHPC‐PDT and also affected satellite glial cell viability, whereas 2‐MeOE2 caused only a small increase in neuron sensitivity (not significant). Pretreatment using a combination of DDC and l‐BSO caused a near total loss of neuron and glial cell viability in treatment and control conditions. These findings suggest that the SOD‐1 and glutathione pathways are likely to be involved in the neuronal sparing associated with mTHPC‐PDT.

Anti-cancer effects of oncolytic viral therapy combined with photodynamic therapy in human pancreatic cancer cell lines.

BACKGROUND Oncolytic viral therapy and photodynamic therapy are potential therapies for inoperable or advanced pancreatic cancer. Our aim was to investigate the anti-cancer killing effects of reovirus therapy combined with protoporphyrin IX (PpIX)-mediated photodynamic therapy on a variety of human pancreatic cancer cell lines. METHODS Pancreatic cancer cell lines (PsPC-1 and BXPC-3) and a non-cancer control cell line (HEK293) were infected with reovirus serotype 3 strain Dearing (T3D) at 0, 0·1, 1, and 10 plaque-forming units (PFU) per cell for 48 h. Cells were incubated with PpIX pro-drug 5-aminolevulinic acid (5-ALA) at 0, 1, 2, 3, and 4 mM for 4 h. Then, cells were photo-irradiated for 15 min with visible red light-emitting diodes with a light-fluence of 0·54 J/cm(2) of 653 nm (PpIX optimal excitation wavelength). The killing effects of reovirus combined with PpIX-mediated photodynamic therapy were analysed in methylthiazoltetrazolium (MTT) and trypan blue assays. The effect of adding reovirus after photodynamic therapy was also assessed. The statistical significance of the difference between groups was assessed with the two-tailed Students t test. p<0·05 was considered statistically significant. FINDINGS Reovirus monotherapy induced cell death in the two pancreatic lines (mean 57% [SE 10·2] at 10 PFU per cell). PpIX-mediated PDT monotherapy induced cell death in a dose-dependent manner (mean 10% [SE 2·2], 30 [6·4], 50 [8·2], and 70 [13·2] after 1, 2, 3, and 4 mM 5-ALA, respectively). Reovirus with PpIX-mediated photodynamic therapy resulted in a significantly increased cytotoxic effect compared with reovirus monotherapy and photodynamic therapy (p=0·042) with 100% cell death observed across pancreatic cell lines with 10 PFU per cell combined with 1 and 2 mM 5-ALA. There was no difference in cytotoxicity observed between added reovirus before or after photodynamic therapy. INTERPRETATION To our knowledge, this is the first in-vitro study to combine reovirus oncolytic viral therapy with PpIX-mediated photodynamic therapy to treat pancreatic cancer. These results show a significant additive effect in cell killing and they provide initial evidence for a novel combined therapeutic intervention. FUNDING National Institute for Health Research.

Assessment of the activity of a novel nociceptin/orphanin FQ analogue at recombinant human nociceptin/orphanin FQ receptors expressed in Chinese hamster ovary cells

The neuropeptide nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the nociceptin receptor (NOP). In an attempt to identify high potency NOP agonists for use in the brain we have compared the activity of a novel N/OFQ analogue [Phe(1)Psi(CH(2)-O)Gly(2)]N/OFQ(1-13)NH(2) ([F/G-O]) with the existing [Phe(1)Psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) ([F/G]). Both peptides are modified between the first two N-terminal amino acids and are further compared with the agonist template N/OFQ(1-13)NH(2) in [(3)H]N/OFQ binding, GTPgamma[(35)S] binding and cAMP inhibition studies using Chinese hamster ovary cells expressing the recombinant human NOP. All peptides displaced [(3)H]N/OFQ, stimulated GTPgamma[(35)S] binding and inhibited cAMP formation. In [(3)H]N/OFQ binding and GTPgamma[(35)S] binding the rank order affinity and potency was N/OFQ(1-13)NH(2)>[F/G-O]>[F/G]. In GTPgamma[(35)S] binding [F/G] was a clear partial agonist with intrinsic activity (E(max) stimulation factor, mean+/-SEM, n=4) of 7.75+/-1.02 compared with N/OFQ(1-13)NH(2) of 11.13+/-1.76. The efficacy of [F/G-O] (10.17+/-1.88) approached that of the full agonist N/OFQ(1-13)NH(2). Downstream, at the level of cAMP formation, all peptides were full agonists with the following rank order potency: N/OFQ(1-13)NH(2)>[F/G-O]=[F/G]. The enhanced potency and intrinsic activity of the novel [F/G-O] modification makes this an interesting peptide for further in vivo analysis.

Treatment of peritoneal carcinomatosis with photodynamic therapy: Systematic review of current evidence

BACKGROUND Peritoneal carcinomatosis results when tumour cells implant and grow within the peritoneal cavity. Treatment and prognosis vary based on the primary cancer. Although therapy with intention-to-cure is offered to selective patients using cytoreductive surgery with chemotherapy, the prognosis remains poor for most of the patients. Photodynamic therapy (PDT) is a cancer-therapeutic modality where a photosensitiser is administered to patients and exerts a cytotoxic effect on cancer cells when excited by light of a specific wavelength. It has potential application in the treatment of peritoneal carcinomatosis. METHODS We systematically reviewed the evidence of using PDT to treat peritoneal carcinomatosis in both animals and humans (Medline/EMBASE searched in June 2017). RESULTS Three human and 25 animal studies were included. Phase I and II human trials using first-generation photosensitisers showed that applying PDT after surgical debulking in patients with peritoneal carcinomatosis is feasible with some clinical benefits. The low tumour-selectivity of the photosensitisers led to significant toxicities mainly capillary leak syndrome and bowel perforation. In animal studies, PDT improved survival by 15-300%, compared to control groups. PDT led to higher tumour necrosis values (categorical values 0-4 [4=highest]: PDT 3.4±1.0 vs. control 0.4±0.6, p<0.05) and reduced tumour size (residual tumour size is 10% of untreated controls, p<0.001). CONCLUSION PDT has potential in treating peritoneal carcinomatosis, but is limited by its narrow therapeutic window and possible serious side effects. Recent improvement in tumour-selectivity and light delivery systems is promising, but further development is needed before PDT can be routinely applied for peritoneal carcinomatosis.