Design and development of miRNA and stem cell based micro/nano systems as lung disease therapy

Patients suffering from lung diseases are often treated with lung transplantation, which is considered a definitive therapy offered for patients with end-stage lung failure. Because of this, it improves survival rates besides boosting the quality of life for lung transplant recipients. However, lung transplantation suffers from a number of flaws, mainly a scarcity of organs and tissue donors. In this thesis, three approaches were attempted to address these issues. These were gene therapy, microfluidic -device -assisted stem cell delivery and tissue engineering. MicroRNA (miRNA) is emerging as a new class of gene therapeutics with the promise of healing many diseases. The applications of miRNA often require miRNA to be transfected efficiently inside cells. Chapter 3 of this work is particularly devoted to investigating the ability of a liquid marble (LM) miniature bioreactor to enhance miRNA transfection. To achieve this, suspended A549 lung cancer spheroids were first generated using liquid marble (LM) by taking advantage of the LM non-adhesive shell, thus promoting the cell-to-cell attachment that is essential for spheroid formation. Next, a tumour suppressor (miR-126) was transfected to spheroids inside LM. It was observed that the resulting miR-126 expression and the subsequent down-regulation in VEGF-A expression reached a level that cannot be reached using conventional methods, such as transfecting miR-126 to monolayer cells or transfecting miR-126 to spheroids generated using liquid suspension methods. There are multiple possible reasons for the greater miRNA expression observed in the LM bioreactor. First of all, miRNA is much smaller than many chemotherapeutic drugs and thus penetrated well into spheroids. Secondly, the PTFE shell possesses ultra-low surface free energy and so does not absorb and waste any miRNA through non-specific adsorption. Lastly, the hydrophobic interface present on the LM shell may play a role in promoting the movement of hydrophilic miRNA towards the centre of the LM where spheroids resided. Overall, this study demonstrates that LM can serve not only as a platform that produces tumour-like spheroids, but also as an efficient microbioreactor vessel that enhances miRNA transfection and outperforms conventional transfection vessels. As part of this approach, a novel micro-centrifugation device was also developed to generate multicellular spheroids, as shown in Chapter 4, these spheroids are useful for lung cancer behaviour and drug screening studies. Intense acoustically driven micro-centrifugation flows were employed to enhance the assembly of multicellular spheroids in the microwells of a tissue culture plate. This ability to interface microfluidics with commonly used tissue culture plasticware is a significant advantage, mainly because it can be multiplied for high-throughput operation and allows the retention of existing analytical equipment designed to fit current laboratory formats. The micro-centrifugation flow induced in microwells coated with a low adhesive hydrogel rapidly enhanced the concentration of BT-474 cells, resulting in tight aggregates within a minute. This was considerably faster than the conventional hanging drop and liquid overlay methods, which typically require a day to maintain their viability. Despite the promise of stem cell therapy for lung therapeutics and repair, there are very few viable means for directly delivering stem cells to locally target the respiratory airways via inhalation. This is not surprising given the significant challenges in aerosolising stem cells, particularly given their susceptibility to damage under the sizable stresses involved in the nebulisation process. The current study presents promising results using a microfluidic acoustic nebulisation platform in Chapter 5. This platform is not only low-cost and portable, but its high MHz order frequencies were effective in preserving the structural and functional integrity of mesenchymal stem cells (MSCs) during the nebulisation process. This was verified through an assessment of the viability, structure, metabolic activity, proliferation ability and genetic constitution of the nebulised MSCs using a variety of assays that included: cell viability staining, flow cytometry, reverse transcription, quantitative polymerase chain reaction and immuno-phenotyping. Given the novelty of inhaled stem cell therapy, comparisons with other delivery methods are difficult at present due to lack of comparable studies. More data for benchmarking are likely to become available in the near future due to the increasing interest in stem cell therapy, given the absence of viable alternative treatment régimes for respiratory ailments to date. Nonetheless, the results in this work provide compelling evidence that the SAW nebulisation platform, with its inherent benefits of low cost and portability, constitutes an attractive tool for the delivery of stem cells via inhalation for the treatment and repair of lung function. In Chapter 6, a novel tissue scaffold composed of enzymatically cross-linked gelatin was developed. Essentially, mESCs were encapsulated in this scaffold simultaneously with the scaffold being cross-linked. A novel injectable hydrogel system composed of gelatin-3,4-dihydroxyhydrocinnamic acid (Gtn-DHHA) conjugates were cross-linked rapidly by laccase-mediated oxidation to form a hydrogel tissue scaffold which could simultaneously immobilise mESCs. This scaffold system supported the proliferation and differentiation of mESCs into lung epithelial cells in both the presence and absence of growth factors. The stiffness of the hydrogels was readily modulated by altering polymer precursor concentration. Gtn-DHHA hydrogel was maintained in air-liquid interface (ALI) conditions to mimic the lung micro- environment and mESC differentiation was evaluated by measuring the expression of E-cadherin and Foxa2, both of which are considered to be definitive endoderm markers. Furthermore, the expression of Pept2, Sema4F, Unc5b, and Ttf1 in hydrogel-encapsulated cells was assessed using the quantitative polymerase reaction (qPCR) and immunofluorescence assays. The expression of these markers was up-regulated in the presence and absence of growth factors, in particular under air-liquid interface by comparison with submerged and control cultures. This scaffold facilitated mESC differentiation into alveolar epithelial cells, even without growth factors, and is therefore a promising approach for lung repair. In summary, this thesis first presents a novel, effective method—namely liquid marble— for better generation of multicellular spheroids to enhance miR-126 PEI nanoparticles transfection. It then introduces a novel method called a SAW device, which is also used to produce multicellular spheroids, and to generate MSC-laden aerosols to cure small lung damage. Finally, it presents a novel three-dimensional (3D) biodegradable scaffold as an alternative material for an extracellular matrix (ECM) for improved stem cell survival, proliferation, and differentiation. This is considered to be a critical step for in-vitro differentiation of ESCs into lung epithelial cells for lung repair and regeneration

Antibiotic use during pregnancy increases offspring asthma severity in a dose‐dependent manner

Background: The use of antibiotics during pregnancy is associated with increased allergic asthma risk in the offspring, and given that approximately 25% of pregnant women are prescribed antibiotics, it is important to understand the mechanisms contributing to this phenomenon. Currently, there are no studies that directly test this association experimentally. Our objective was to develop a mouse model in which antibiotic treatment during pregnancy results in increased offspring asthma susceptibility. Methods: Pregnant mice were treated daily from gestation day 8-17 with an oral solution of the antibiotic vancomycin, and three concentrations were tested. At weaning, offspring were subjected to an adjuvant-free experimental asthma protocol using ovalbumin as an allergen. The composition of the gut microbiome was determined in mothers and offspring with samples collected from five different time points; shortchain fatty acids were also analyzed in allergic offspring. Results: We found that maternal antibiotic treatment during pregnancy was associated with increased offspring asthma severity in a dose-dependent manner. Furthermore, maternal vancomycin treatment during pregnancy caused marked changes in the gut microbiome composition in both mothers and pups at several different time points. The increased asthma severity and intestinal microbiome changes in pups were also associated with significantly decreased cecal short-chain fatty acid concentrations. Conclusion: Consistent with the "Developmental Origins Hypothesis," our results confirm that exposure to antibiotics during pregnancy shapes the neonatal intestinal environment and increases offspring allergic lung inflammation

Neighborhood Characteristics and the Mental Health of Caregivers Cohabiting With Care Recipients Diagnosed With Alzheimer’s Disease

While studies have documented the influence of caregiver and care recipient factors on caregiver health, it is important to address the potential impact of neighborhood contexts. This study estimated the cross-sectional associations between neighborhood characteristics and mental health among caregivers cohabiting with Alzheimer’s disease care recipients that were experiencing severe or non-severe neuropsychiatric symptoms (NPSs) (e.g., aggression/anxiety). We obtained data collected in 2010 on caregivers and care recipients (n While studies have documented the influence of caregiver and care recipient factors on caregiver health, it is important to address the potential impact of neighborhood contexts. This study estimated the cross-sectional associations between neighborhood characteristics and mental health among caregivers cohabiting with Alzheimer’s disease care recipients that were experiencing severe or non-severe neuropsychiatric symptoms (NPSs) (e.g., aggression/anxiety). We obtained data collected in 2010 on caregivers and care recipients (n = 212) from a subset of South Carolina’s Alzheimer’s Disease Registry. Neighborhood measures (within 1 mile of the residence) came from the American Community Survey and the Rural-Urban Commuting Area Code. We categorized the neighborhood median household income into tertiles, namely, “low” (\u3c$31,000), “medium” ($31,000–40,758), and “high” (\u3e$40,758), and rurality as “large urban,” “small urban,” and “rural.” We used negative binomial regression to estimate the prevalence ratios (PRs) and 95% confidence intervals (CIs) for caregiver mental health using neighborhood characteristics. The mean age was 58 ± 10.3 years, 85% were women, and 55% were non-Hispanic Black. Among the caregivers cohabiting with a recipient experiencing severe NPS, higher distress was experienced by caregivers living in low- (PR = 1.61 (95% CI = 1.26–2.04)) and medium- (PR = 1.45 (95% CI = 1.17–1.78)) vs. high-income neighborhoods after an adjustment. These results suggest that neighborhood characteristics may amplify other social stressors experienced by caregivers

A baseline survey of potentially toxic elements in the soil of north-west Syria following a decade of conflict

We present the first region-wide chemical survey of soils in NW Syria following more than a decade of ongoing conflict. We sampled topsoil at 66 sites, typically located in marginal agricultural (orchards, arable) or peri-urban settings, grouped around 21 localities covering the whole area of NW Syria currently under Syrian Opposition control. Samples were analysed in the UK using ICP-MS and ICP-OES. Topsoil total concentrations of heavy metals are broadly consistent with pre-war data from Aleppo and recent data from nearby Turkey. Principal Components Analysis (PCA) of associations among the sampling sites identified three groupings. Ni (133.30 ± 72.12 mg/kg) and Cr (122.14 ± 52.25 mg/kg) exist in all samples at levels in excess of typical European guideline thresholds for agricultural soil. Observed Cd (0.57 ± 0.93 mg/kg), Co (23.07 ± 18.48 mg/kg) and As (6.65 ± 4.51 mg/kg) concentrations are up to three times comparable values from nearby agricultural regions in southern Turkey. Maximum observed values for Cd, As, and Co, which exceed EU thresholds, are concentrated in a corridor around Sarmada to the west of Aleppo which has seen some of the most intense conflict-related impacts. Cu (28.33 ± 17.11 mg/kg), Pb (15.65 ± 10.85 mg/kg) and Zn (73.64 ± 40.15 mg/kg) also observe maxima in the Sarmada corridor, but show a more even distribution across the region, widely at values above comparable regional values for agriculture but below EU threshold concentrations. We interpret the occurrence of Ni-Cr as consistent with intensive agriculture using wastewater-contaminated irrigation and fertilisers. Cd-As-Co and Cu-Pb-Zn are likely anthropogenic and reflect intense pressures of conflict, informal settlement, unregulated industry and untreated wastewater irrigation on a historically agricultural region. The sampling method was designed to capture regional variations from a minimal dataset and it is likely that local topsoil concentrations at specific points of impact (proximal to locations of shelling, industry, effluent release or population) will be considerably higher than those reported here. This study establishes an important baseline reference for further targeted studies to identify and mitigate specific pollution hazards in this region of ongoing, extreme humanitarian and ecological threat

Inhibition of Cell Proliferation and MAP Kinase and Akt Pathways in Oral Squamous cell Carcinoma by Genistein and Biochanin A

High morbidity and mortality associated with oral squamous cell carcinoma (OSCC) are largely attributable to late stage diagnosis. Despite significant advances in therapeutic strategies, the five-year survival rate for oral cancer remains at about 50%. A chemopreventive approach may be an effective alternative or adjunct to current therapies. Previous studies have shown anti-tumor effects of isoflavones in several cancers, including oral cancer. However, their mechanisms of action are still unclear. We hypothesized that isoflavones inhibit multiple signaling pathways implicated in oral carcinogenesis. To address our hypothesis, we investigated the effects of three isoflavone derivatives, genistein, biochanin A and daidzein, on SCC15 and SCC25 squamous cell carcinoma cell lines. In cell proliferation experiments, we found that genistein and biochanin A inhibited SCC15 and SCC25 cell growth with an IC50 of 50 μM. We also investigated the effect of isoflavones on ERK and Akt pathways. Our results, from western blot analysis, suggest that both genistein and biochanin A induced decreases in phosphorylation of ERK and Akt at treatment concentrations of 20, 50 and 100 μM. Taken together, our results clearly demonstrate a differential regulation of signaling pathways by various isoflavones in OSCC cell lines. Thus, tumor progression models can be utilized to study the preventive and therapeutic roles of isoflavones in oral cancer cell lines

Sulfatase-2 from Cancer Associated Fibroblasts: An Environmental Target for Hepatocellular Carcinoma?

Introduction: Heparin sulphate proteoglycans in the liver tumour microenvironment (TME) are key regulators of cell signalling, modulated by sulfatase-2 (SULF2). SULF2 overexpression occurs in hepatocellular carcinoma (HCC). Our aims were to define the nature and impact of SULF2 in the HCC TME. Methods: In liver biopsies from 60 patients with HCC, expression and localization of SULF2 were analysed associated with clinical parameters and outcome. Functional and mechanistic impacts were assessed with immunohistochemistry (IHC), in silico using The Cancer Genome Atlas (TGCA), in primary isolated cancer activated fibroblasts, in monocultures, in 3D spheroids, and in an independent cohort of 20 patients referred for sorafenib. IHC targets included αSMA, glypican-3, β-catenin, RelA-P-ser536, CD4, CD8, CD66b, CD45, CD68, and CD163. SULF2 impact of peripheral blood mononuclear cells was assessed by migration assays, with characterization of immune cell phenotype using fluorescent activated cell sorting. Results: We report that while SULF2 was expressed in tumour cells in 15% (9/60) of cases, associated with advanced tumour stage and type 2 diabetes, SULF2 was more commonly expressed in cancer-associated fibroblasts (CAFs) (52%) and independently associated with shorter survival (7.2 vs. 29.2 months, p = 0.003). Stromal SULF2 modulated glypican-3/β-catenin signalling in vitro, although in vivo associations suggested additional mechanisms underlying the CAF-SULF2 impact on prognosis. Stromal SULF2 was released by CAFS isolated from human HCC. It was induced by TGFβ1, promoted HCC proliferation and sorafenib resistance, with CAF-SULF2 linked to TGFβ1 and immune exhaustion in TGCA HCC patients. Autocrine activation of PDGFRβ/STAT3 signalling was evident in stromal cells, with the release of the potent monocyte/macrophage chemoattractant CCL2 in vitro. In human PBMCs, SULF2 preferentially induced the migration of macrophage precursors (monocytes), inducing a phenotypic change consistent with immune exhaustion. In human HCC tissues, CAF-SULF2 was associated with increased macrophage recruitment, with tumouroid studies showing stromal-derived SULF2-induced paracrine activation of the IKKβ/NF-κB pathway, tumour cell proliferation, invasion, and sorafenib resistance. Conclusion: SULF2 derived from CAFs modulates glypican-3/β-catenin signalling but also the HCC immune TME, associated with tumour progression and therapy resistance via activation of the TAK1/IKKβ/NF-κB pathway. It is an attractive target for combination therapies for patients with HCC

Inhibition of cancer cell invasion and metastasis by genistein

Genistein is a small, biologically active flavonoid that is found in high amounts in soy. This important compound possesses a wide variety of biological activities, but it is best known for its ability to inhibit cancer progression. In particular, genistein has emerged as an important inhibitor of cancer metastasis. Consumption of genistein in the diet has been linked to decreased rates of metastatic cancer in a number of population-based studies. Extensive investigations have been performed to determine the molecular mechanisms underlying genistein’s antimetastatic activity, with results indicating that this small molecule has significant inhibitory activity at nearly every step of the metastatic cascade. Reports have demonstrated that, at high concentrations, genistein can inhibit several proteins involved with primary tumor growth and apoptosis, including the cyclin class of cell cycle regulators and the Akt family of proteins. At lower concentrations that are similar to those achieved through dietary consumption, genistein can inhibit the prometastatic processes of cancer cell detachment, migration, and invasion through a variety of mechanisms, including the transforming growth factor (TGF)-β signaling pathway. Several in vitro findings have been corroborated in both in vivo animal studies and in early-phase human clinical trials, demonstrating that genistein can both inhibit human cancer metastasis and also modulate markers of metastatic potential in humans, respectively. Herein, we discuss the variety of mechanisms by which genistein regulates individual steps of the metastatic cascade and highlight the potential of this natural product as a promising therapeutic inhibitor of metastasis