A novel non-viral delivery method that enables efficient engineering of primary human T cells for ex vivo cell therapy applications.

Background Aims: Next-generation immune cell therapy products will require complex modifications using engineering technologies that can maintain high levels of cell functionality. Non-viral engineering methods have the potential to address limitations associated with viral vectors. However, while electroporation is the most widely used non-viral modality, concerns about its effects on cell functionality have led to the exploration of alternative approaches.

Delivery of nucleic acids to ex vivo porcine airways using electrospray.

Aim Of The Study: Nucleic acid-based therapies have the potential to provide clinically meaningful benefit across a wide spectrum of lung disease. However, in vivo delivery remains a challenge. Here we examined the feasibility of using electrospray to deliver nucleic acids to both porcine tracheal tissue sections and whole lung ex vivo.

Vector-free intracellular delivery by reversible permeabilization.

Despite advances in intracellular delivery technologies, efficient methods are still required that are vector-free, can address a wide range of cargo types and can be applied to cells that are difficult to transfect whilst maintaining cell viability. We have developed a novel vector-free method that uses reversible permeabilization to achieve rapid intracellular delivery of cargos with varying composition, properties and size. A permeabilizing delivery solution was developed that contains a low level of ethanol as the permeabilizing agent.

The development of a tissue-engineered tracheobronchial epithelial model using a bilayered collagen-hyaluronate scaffold.

Today, chronic respiratory disease is one of the leading causes of mortality globally. Epithelial dysfunction can play a central role in its pathophysiology. The development of physiologically-representative in vitro model systems using tissue-engineered constructs might improve our understanding of epithelial tissue and disease.

SMAD Signaling in the Airways of Healthy Rhesus Macaques versus Rhesus Macaques with Asthma Highlights a Relationship Between Inflammation and Bone Morphogenetic Proteins.

Bone morphogenetic protein (BMP) signaling is important for correct lung morphogenesis, and there is evidence of BMP signaling reactivation in lung diseases. However, little is known about BMP signaling patterns in healthy airway homeostasis and inflammatory airway disease and during epithelial repair. In this study, a rhesus macaque (Macaca mulatta) model of allergic airway disease was used to investigate BMP signaling throughout the airways in health, disease, and regeneration.

Respiratory Tissue Engineering: Current Status and Opportunities for the Future.

Currently, lung disease and major airway trauma constitute a major global healthcare burden with limited treatment options. Airway diseases such as chronic obstructive pulmonary disease and cystic fibrosis have been identified as the fifth highest cause of mortality worldwide and are estimated to rise to fourth place by 2030. Alternate approaches and therapeutic modalities are urgently needed to improve clinical outcomes for chronic lung disease.

Anatomy and bronchoscopy of the porcine lung. A model for translational respiratory medicine.

The porcine model has contributed significantly to biomedical research over many decades. The similar size and anatomy of pig and human organs make this model particularly beneficial for translational research in areas such as medical device development, therapeutics and xenotransplantation. In recent years, a major limitation with the porcine model was overcome with the successful generation of gene-targeted pigs and the publication of the pig genome.

Regulation of epithelial to mesenchymal transition by bone morphogenetic proteins.

Epithelial to mesenchymal transition (EMT) is a process in which fully differentiated epithelial cells lose many of their epithelial characteristics and adopt features typical of mesenchymal cells, thus allowing cells to become migratory and invasive. EMT is a critical process in development and its role in cancer and fibrosis is becoming increasingly recognised. It is also becoming apparent that EMT is not just restricted to embryonic development and disease in adults, but in fact may be an important process for the maintenance and regeneration of adult tissue architecture.

IFN-γ stimulated human umbilical-tissue-derived cells potently suppress NK activation and resist NK-mediated cytotoxicity in vitro.

Umbilical cord tissue represents a unique source of cells with potential for cell therapy applications for multiple diseases. Human umbilical tissue-derived cells (hUTC) are a developmentally early stage, homogenous population of cells that are HLA-ABC dim, HLA-DR negative, and lack expression of co-stimulatory molecules in the unactivated state. The lack of HLA-DR and co-stimulatory molecule expression on unactivated hUTC may account for their reduced immunogenicity, facilitating their use in allogeneic settings.

Bone morphogenetic proteins enhance an epithelial-mesenchymal transition in normal airway epithelial cells during restitution of a disrupted epithelium.

Background: Mechanisms of airway repair are poorly understood. It has been proposed that, following injury, progenitor populations such as club cells (Clara) become undifferentiated, proliferate and re-differentiate to re-epithelialise the airway. The exact phenotype of such cells during repair is unknown however.

Olfactomedin III expression contributes to anoikis-resistance in clonal variants of a human lung squamous carcinoma cell line.

Three clonal subpopulations of DLKP, a poorly differentiated squamous lung carcinoma cell line, display striking differences in ability to survive in suspension (anoikis resistance). DLKP-SQ is anoikis resistant (7.5% anoikis at 24 h).

Bone morphogenetic protein signalling in airway epithelial cells during regeneration.

Mechanisms of lung regeneration after injury remain poorly understood. Bone morphogenetic protein 4 (BMP4) is critical for lung morphogenesis and regulates differentiation of the airway epithelium during development, although its mechanism of action is unknown. The role of BMPs in adult lungs is unclear.

Regulation of surfactant protein B gene expression in bone marrow-derived cells.

While investigating the differentiation potential of bone marrow-derived cells, we previously demonstrated upregulated expression of the lung-related surfactant protein B (SP-B) gene in hematopoietic progenitor cells (HPCs) when they were cocultured with macerated lung tissue. During coculture, HPCs differentiated toward a dendritic-like myeloid cell phenotype (hematopoietic progenitor cell-derived dendritic-like cells [HPC-DCs]). However, immature dendritic cells (iDCs) cocultured under identical conditions did not express SP-B mRNA before or after coculture.

A possible role for protein synthesis, extracellular signal-regulated kinase, and brain-derived neurotrophic factor in long-term spatial memory retention in the water maze.

Hippocampal protein synthesis is dependent upon a number of different molecular and cellular mechanisms that act together to make previously labile memories more stable and resistant to disruption. Both brain-derived neurotrophic factor (BDNF) and extracellular signal-regulated kinase (ERK) are known to play an important role in protein synthesis-dependent memory consolidation, via the mitogen-activated protein-kinase (MAP-K) signaling pathway during the transcription phase of protein synthesis. The current study investigates the influence of protein synthesis inhibition (PSI) by cycloheximide on spatial learning and memory.

Comparative evaluation of viral, nonviral and physical methods of gene delivery to normal and transformed lung epithelial cells.

Few studies have directly compared the efficiencies of gene delivery methods that target normal lung cells versus lung tumor cells. We report the first study directly comparing the efficiency and toxicity of viral [adeno-associated virus (AAV2, 5, 6) and lentivirus], nonviral (Effectene, SuperFect and Lipofectamine 2000) and physical [particle-mediated gene transfer (PMGT)] methods of gene delivery in normal mouse lung cells and in mouse adenocarcinoma cells. Lentivirus pseudotyped with the vesicular stomatitis virus glycoprotein was the most efficient gene transfer method for normal mouse airway epithelial cells [25.

Reducing the false positive rate in the non-parametric analysis of molecular coevolution.

Background: The strength of selective constraints operating on amino acid sites of proteins has a multifactorial nature. In fact, amino acid sites within proteins coevolve due to their functional and/or structural relationships. Different methods have been developed that attempt to account for the evolutionary dependencies between amino acid sites.

BMP4 induces an epithelial-mesenchymal transition-like response in adult airway epithelial cells.

Bone morphogenetic proteins (BMPs) are critical morphogens and play key roles in epithelial-mesenchymal transitions (EMTs) during embryogenesis. BMP4 is required for early mesoderm formation and also regulates morphogenesis and epithelial cell differentiation in developing lungs. While, BMP signalling pathways are activated during lung inflammation in adult mice, the role of BMPs in adult lungs remains unclear.

5-Bromo-2-deoxyuridine activates DNA damage signalling responses and induces a senescence-like phenotype in p16-null lung cancer cells.

5-Bromo-2-deoxyuridine (BrdU) is a thymidine analogue that is incorporated into replicating DNA. Although originally designed as a chemotherapeutic agent, sublethal concentrations of BrdU have long been known to alter the growth and phenotype of a wide range of cell types. Mechanisms underlying these BrdU-mediated effects remain unknown, however.

Posttranslational truncation of E-cadherin and significance for tumour progression.

Stable intraepithelial adhesion complexes are essential for the maintenance of epithelial integrity. Alterations in these complexes are key events in the development and progression of many diseases. One of the major proteins involved in maintaining epithelial cell-cell adhesion is the cell-adhesion junction protein E-cadherin, a member of the cadherin family of transmembrane adhesion proteins.

p21(Waf1/Cip1) regulates proliferation and apoptosis in airway epithelial cells and alternative forms have altered binding activities.

p21(Waf1/Cip1) plays central roles in proliferation, differentiation, and apoptosis. Alterations in the expression and subcellular localisation of p21 occur during several lung diseases but the roles of p21 in the lung epithelium are unknown. The effects of p21 on proliferation and apoptosis in mouse airway epithelial cells (AECs) were examined using p21-null mice.

Absence of p53 in Clara cells favours multinucleation and loss of cell cycle arrest.

Background: The p53 oncosuppressor protein is a critical mediator of the response to injury in mammalian cells and is mutationally inactivated in the majority of lung malignancies. In this analysis, the effects of p53-deficiency were investigated in short-term primary cultures of murine bronchiolar Clara cells. Clara cells, isolated from gene-targeted p53-deficient mice, were compared to cells derived from wild type littermates.