Paladd Asavarut

The role of HMGB1 in inflammation-mediated organ injury.

HMGB1 is a chromosome-binding protein that also acts as a damage-associated molecular pattern molecule. It has potent proinflammatory effects and is one of key mediators of organ injury. Evidence from research has revealed its involvement in the signaling mechanisms of Toll-like receptors and the receptor for advanced glycation end-products in organ injury. HMGB1-mediated organ injuries are acute damage including ischemic, mechanical, allograft rejection and toxicity, and chronic diseases of the heart, kidneys, lungs, and brain. Strategies against HMGB1 and its associated cellular signal pathways need to be developed and may have preventive and therapeutic potentials in organ injury.

Left Cathodal Trans-Cranial Direct Current Stimulation of the Parietal Cortex Leads to an Asymmetrical Modulation of the Vestibular-Ocular Reflex

Multi-sensory visuo-vestibular cortical areas within the parietal lobe are important for spatial orientation and possibly for descending modulation of the vestibular-ocular reflex (VOR). Functional imaging and lesion studies suggest that vestibular cortical processing is localized primarily in the non-dominant parietal lobe. However, the role of inter-hemispheric parietal balance in vestibular processing is poorly understood. Therefore, we tested whether experimentally induced asymmetries in right versus left parietal excitability would modulate vestibular function. VOR function was assessed in right-handed normal subjects during caloric ear irrigation (30 °C), before and after trans-cranial direct current stimulation (tDCS) was applied bilaterally over the parietal cortex. Bilateral tDCS with the anode over the right and the cathode over the left parietal region resulted in significant asymmetrical modulation of the VOR, with highly suppressed responses during the right caloric irrigation (i.e. rightward slow phase nystagmus). In contrast, we observed no VOR modulation during either cathodal stimulation of the right parietal cortex or SHAM tDCS conditions. Application of unilateral tDCS revealed that the left cathodal stimulation was critical in inducing the observed modulation of the VOR. We show that disruption of parietal inter-hemispheric balance can induce asymmetries in vestibular function. This is the first report using neuromodulation to show right hemisphere dominance for vestibular cortical processing.

Bidirectional Modulation of Numerical Magnitude

Numerical cognition is critical for modern life; however, the precise neural mechanisms underpinning numerical magnitude allocation in humans remain obscure. Based upon previous reports demonstrating the close behavioral and neuro-anatomical relationship between number allocation and spatial attention, we hypothesized that these systems would be subject to similar control mechanisms, namely dynamic interhemispheric competition. We employed a physiological paradigm, combining visual and vestibular stimulation, to induce interhemispheric conflict and subsequent unihemispheric inhibition, as confirmed by transcranial direct current stimulation (tDCS). This allowed us to demonstrate the first systematic bidirectional modulation of numerical magnitude toward either higher or lower numbers, independently of either eye movements or spatial attention mediated biases. We incorporated both our findings and those from the most widely accepted theoretical framework for numerical cognition to present a novel unifying computational model that describes how numerical magnitude allocation is subject to dynamic interhemispheric competition. That is, numerical allocation is continually updated in a contextual manner based upon relative magnitude, with the right hemisphere responsible for smaller magnitudes and the left hemisphere for larger magnitudes.

Separate attentional components modulate early visual cortex excitability.

Disruption of the right lateralised fronto-parietal attentional network using neuro-modulation techniques has been shown to induce both functional and perceptual modulation of early visual cortex (Silvanto, Muggleton, Lavie, & Walsh, 2009). Such modulation is suggested to be mediated by interhemispheric competition (Silvanto et al., 2009). To date in neurologically normal subjects no behavioural demonstration of such modulation exists. In this study, we stimulated the vestibular system duringperformanceof anattentional task. Aprevious studyhas demonstrated that passive rotation combined with performance of a visual attentional task results in asymmetric modulation of the brainstemmediated vestibulo-ocular reflex (VOR) (Arshad, Nigmatullina, & Bronstein, 2013). The modulation of the VOR is suggested to occur as a result of activating overlapping cortical networks responsible for processing both vestibular information and the attentional task in the right parietal lobe (Corbetta & Shulman, 2002; Dieterich et al., 2003; Miller et al., 2000; Van Elk & Blanke, 2012), resulting in inhibition of the left hemisphere via interhemispheric competition (Arshad et al., 2013; Miller et al., 2000). This hypothesis was directly tested in a recent study where transcranial direct current stimulation of the parietal cortex was employed to assess the effect upon the VOR (Arshad, Nigmatullina, Roberts et al., 2013), with the largest modulation of the VOR observed during cathodal stimulation of left parietal cortex. Thus in this study, we combined caloric stimulationwith a visual attention task to disrupt parietal interhemispheric balance in normal subjects, and measured the possible effect on V1/V2 excitability. Moreover, for the first time we delineate the specific contributions of spatial versus non-spatial attentional networks in modulating early visual cortex. We assessed V1 excitability using phosphenes, elicited by briefly stimulating the visual cortex using single pulse transcranial magnetic stimulation (TMS), with the intensity required to elicit a phosphene reflecting the underlying cortical excitability (Marg & Rudiak, 1994). Eighteen righthanded participants (male 14, mean age 1⁄4 24; range 20e33) gave written informed consent as approved by the local research ethics committee. Subjects were blindfolded and were seated on a chair fitted with a fixed magnetic coil and head restraint system. The head was inclined 30 from the horizontal plane for maximal horizontal canal caloric stimulation. Firstly, V1 stimulation site was localised using a functional method by placing the coil centrally over the inion then moving it dorsally until the brightest stationary phosphene percept is observed in the centre of the visual field (Walsh, Pascual-Leone, & Kosslyn, 2003). Secondly, the threshold was established according to a modified binary staircase algorithm (Tyrrell & Owens, 1988) previously described (Seemungal et al., 2013). Subjects were trained to rate the intensity of the perceived phosphene on a scale from 0 (no phosphene, below threshold) to 5 (maximumbrightness, 100% maximum stimulator output). We then used the established clinical approach for coldwater (30 C) caloric irrigation (left or right ear, randomised order) for 40 sec to activate the vestibular system. The irrigations were separated by a period of 5 minutes to allow for any after effects to subside. Following each irrigation we immediately measured visual cortical excitability using 20 single TMS pulses (GuzmanLopez, Silvanto, & Seemungal, 2011) applied at 20% above phosphene threshold for each individual, with each pulse separated by 3 sec. Subjects responded verbally and rated

Modulation of extracellular matrix in cancer is associated with enhanced tumor cell targeting by bacteriophage vectors

BackgroundGene therapy has been an attractive paradigm for cancer treatment. However, cancer gene therapy has been challenged by the inherent limitation of vectors that are able to deliver therapeutic genes to tumors specifically and efficiently following systemic administration. Bacteriophage (phage) are viruses that have shown promise for targeted systemic gene delivery. Yet, they are considered poor vectors for gene transfer. Recently, we generated a tumor-targeted phage named adeno-associated virus/phage (AAVP), which is a filamentous phage particle whose genome contains the adeno-associated virus genome. Its effectiveness in delivering therapeutic genes to tumors specifically both in vitro and in vivo has been shown in numerous studies. Despite being a clinically useful vector, a multitude of barriers impede gene transduction to tumor cells. We hypothesized that one such factor is the tumor extracellular matrix (ECM).MethodsWe used a number of tumor cell lines from different species and histological types in 2D monolayers or 3D multicellular tumor spheroid (MCTS) models. To assess whether the ECM is a barrier to tumor cell targeting by AAVP, we depleted the ECM using collagenase, hyaluronidase, or combination of both. We employed multiple techniques to investigate and quantify the effect of ECM depletion on ECM composition (including collagen type I, hyaluronic acid, fibronectin and laminin), and how AAVP adsorption, internalisation, gene expression and therapeutic efficacy are subsequently affected. Data were analyzed using a student’s t test when comparing two groups or one-way ANOVA and post hoc Tukey tests when using more than two groups.ResultsWe demonstrate that collagenase and hyaluronidase-mediated degradation of tumor ECM affects the composition of collagen, hyaluronic acid and fibronectin. Consequently, AAVP diffusion, internalisation, gene expression and tumor cell killing were enhanced after enzymatic treatment. Our data suggest that enhancement of gene transfer by the AAVP is solely attributed to ECM depletion. We provide substantial evidence that ECM modulation is relevant in clinically applicable settings by using 3D MCTS, which simulates in vivo environments more accurately.ConclusionOur findings suggest that ECM depletion is an effective strategy to enhance the efficiency of viral vector-guided gene therapy.

Diet and disease: transgressing boundaries between science and society—understanding neglected diseases through the lens of cultural studies and anthropology

It is vital that we consider human health from all perspectives, including the social, geopolitical and cultural aspects of wider society. A prime example of how such forces complicate patterns of disease is provided by examining the underlying epidemiology of cholangiocarcinoma (bile duct cancer (CCA)) in Thailand. With high prevalence in the northeast of Thailand ( Isan ) and most rural communities along the Mekong River in Southeast Asia, CCA in this region of the world results from a neglected tropical disease, chronic liver fluke infection, caused by consuming raw or undercooked freshwater fish infected with Opisthorchis viverrini (liver fluke) sensu lato. Although the relationship between diet and disease is common knowledge in the general population along the Mekong River, the cultural and sociological facets of dietary practice point to challenges that cannot be addressed by science alone. Untangling the CCA epidemic from the complex human behaviour of wanting to eat ‘forbidden’ food in Thailand provides a compelling case of how partnership between social and medical science and the humanities is key to making a sustainable impact in reducing disease patterns in the developing world. While uncooked dishes are distinctive to Isan cuisine, the most infamous cause of CCA in Thailand is the consumption of an uncooked fish dish known as koi pla . It is, however, often overlooked that a large number of Isan staple foods feature fermented raw fish ( pla som ) or use it as a key seasoning ingredient ( pla ra ). The scope of the problem therefore goes beyond a single food item; rather, it is the local diet at large that is responsible for chronic liver fluke infestations and, ultimately, CCA. As a result, the Isan region, in general, and the major constituent province of Khon Kaen, in particular, is home to ca. 80% of the 10 million people …

Chimeric adeno-associated virus and bacteriophage: a potential targeted gene therapy vector for malignant glioma

The incipient development of gene therapy for cancer has fuelled its progression from bench to bedside in mere decades. Of all malignancies that exist, gliomas are the largest class of brain tumors, and are renowned for their aggressiveness and resistance to therapy. In order for gene therapy to achieve clinical success, a multitude of barriers ranging from glioma tumor physiology to vector biology must be overcome. Many viral gene delivery systems have been subjected to clinical investigation; however, with highly limited success. In this review, the current progress and challenges of gene therapy for malignant glioma are discussed. Moreover, we highlight the hybrid adeno-associated virus and bacteriophage vector as a potential candidate for targeted gene delivery to brain tumors.

Thermoresponsive Bacteriophage Nanocarrier as a Gene Delivery Vector Targeted to the Gastrointestinal Tract

The use of the gastrointestinal tract as a site for the local delivery of DNA is an exciting prospect. In order to obtain an effective vector capable of delivering a gene of interest to target cells to achieve sufficient and sustained transgene expression, with minimal toxicity, we developed a new generation of filamentous bacteriophage. This particular bacteriophage was genetically engineered to display an arginine-glycine-aspartic acid (RGD) motif (an integrin-binding peptide) on the major coat protein pVIII and carry a mammalian DNA cassette. One unanticipated observation is the thermoresponsive behavior of engineered bacteriophage. This finding has led us to simplify the isolation method to purify bacteriophage particles from cell culture supernatant by low-temperature precipitation. Our results showed that, in contrast to non-surface modified, the RGD-modified bacteriophage was successfully used to deliver a transgene to mammalian cells. Our in vitro model of the human intestinal follicle-associated epithelium also demonstrated that bacteriophage particles were stable in simulated gastrointestinal fluids and able to cross the human intestinal barrier. In addition, we confirmed an adjuvant property of the engineered bacteriophage to induce nitric oxide production by macrophages. In conclusion, our study demonstrated the possibility of using bacteriophage for gene transfer in the gastrointestinal tract.

672. Hybrid AAV/Phage Vector Enhances Chemotherapy Efficacy Against Cancer

Chemotherapy has been widely used for cancer treatment, both in early and late stages. However, chemotherapy does not selectively target tumor cells, as normal cells are also harmed by the drugs resulting in several side effects. Another major obstacle to the success of chemotherapy in cancer treatment is the development of tumor drug resistance by cancer cells. In order to avoid these problems, the combination of chemotherapy with other therapeutic strategies has been used in order to lower the chemotherapeutic drug dose. Gene therapy is one of the therapeutic strategies that can be combined with chemotherapy. Mammalian viruses are well recognized vehicles for gene therapy, but major drawbacks of these viral vectors are broad tissue tropism following systemic administration (low specificity for the target cells/tissues), their fragility to harsh environments and difficulty for large scale production. Therefore, our group has developed a vector from bacteriophage, a bacteria virus also named phage. This novel engineered phage, called AAV/Phage, displays the RGD4C peptide to target a specific receptor (αvβ3 integrin) on cancer cell surface, while the phage genome is merged with recombinant rAAV2 virus genome carrying the transgene to deliver. In comparison with mammalian viral vectors, the production of AAV/Phage is quicker, simpler and more economical. In addition, the vector is stable at 4oC for many years. The AAV/Phage vector efficiently targeted, delivered, and expressed transgene in cancer cells in vitro. We also proved that the vector selectively targeted gene delivery to tumors after intravenous injection in animal models of cancer. Moreover, in brain tumor models, the vector can penetrate through blood brain barrier and selectively delivers transgene expression to brain tumors. We used our vector as combination therapies with some well-known cancer drugs, such as doxorubicin, and temozolomide. Combination treatment of AAV/Phage vector carrying the Herpes Simplex virus thymidine kinase gene (AAV/Phage-HSVtk.) with doxorubicin increased the targeted cancer cell killing in 2D tissue cultures and 3D tumor spheroids of rat gliosarcoma (9L) and human melanoma (M21) cells. We found that this increase in tumor cell killing was associated with a synergistic effect of doxorubicin on enhancing gene expression by AAV/Phage. We then combined AAV/Phage carrying short hairpin RNA to suppress mTOR gene expression (AAV/Phage-shmTOR) with temozolomide to treat medulloblastoma cells (DAOY). The results exhibited that treatment of medulloblastma with the vector alone efficiently suppresses the expression of mTOR gene, but has no effect on cell killing. Treatment of temozolomide at low dose (500 uM) did not have effect on cell killing, but combination therapies of temozolomide with AAV/Phage-shmTOR significantly increased cell death. Altogether, our results demonstrate that combination of AAV/Phage carrying therapeutic genes with cancer chemotherapeutic drugs is an effective strategy for cancer treatment. In future work, we plan to investigate the efficacy of AAV/Phage and cancer drug combination treatment in pre-clinical models of cancer.