On dendrites in Down syndrome and DS murine models: a spiny way to learn

Since the discovery in the 1970s that dendritic abnormalities in cortical pyramidal neurons are the most consistent pathologic correlate of mental retardation, research has focused on how dendritic alterations are related to reduced intellectual ability. Due in part to obvious ethical problems and in part to the lack of fruitful methods to study neuronal circuitry in the human cortex, there is little data about the microanatomical contribution to mental retardation. The recent identification of the genetic bases of some mental retardation associated alterations, coupled with the technology to create transgenic animal models and the introduction of powerful sophisticated tools in the field of microanatomy, has led to a growth in the studies of the alterations of pyramidal cell morphology in these disorders. Studies of individuals with Down syndrome, the most frequent genetic disorder leading to mental retardation, allow the analysis of the relationships between cognition, genotype and brain microanatomy. In Down syndrome the crucial question is to define the mechanisms by which an excess of normal gene products, in interaction with the environment, directs and constrains neural maturation, and how this abnormal development translates into cognition and behaviour. In the present article we discuss mainly Down syndrome-associated dendritic abnormalities and plasticity and the role of animal models in these studies. We believe that through the further development of such approaches, the study of the microanatomical substrates of mental retardation will contribute significantly to our understanding of the mechanisms underlying human brain disorders associated with mental retardation. (C) 2004 Elsevier Ltd. All rights reserved.

Sunscreen penetration of human skin and related keratinocyte toxicity after topical application

Sunscreen skin penetration and safety assessment should be considered together in order to ensure that in vitro cytotoxicity studies examine relevant doses of these organic chemical UV filters to which viable epidermal cells are realistically exposed. In this study, we sought to determine whether sufficient topically applied sunscreens penetrated into human viable epidermis to put the local keratinocyte cell populations at risk of toxicity. The penetration and retention of five commonly used sunscreen agents ( avobenzone, octinoxate, octocrylene, oxybenzone and padimate O) in human skin was evaluated after application in mineral oil to isolated human epidermal membranes. Sunscreen concentration - human keratinocyte culture response curves were then defined using changes in cell morphology and proliferation ( DNA synthesis using radiolabelled thymidine uptake studies) as evidence of sunscreens causing toxicity. Following 24 h of human epidermal exposure to sunscreens, detectable amounts of all sunscreens were present in the stratum corneum and viable epidermis, with epidermal penetration most evident with oxybenzone. The concentrations of each sunscreen found in human viable epidermis after topical application, adjusting for skin partitioning and binding effects, were at least 5-fold lower, based on levels detected in viable epidermal cells, than those appearing to cause toxicity in cultured human keratinocytes. It is concluded that the human viable epidermal levels of sunscreens are too low to cause any significant toxicity to the underlying human keratinocytes. Copyright (C) 2005 S. Karger AG, Basel.

In vitro bioactivity of MOEP grafted ePTFE membranes for craniofacial applications

The bioactivity of three methacryloyloxyethyl phosphate (MOEP) grafted expanded polytetrafluoroethylene (ePTFE) membranes with varying surface coverage as well as unmodified ePTFE was investigated through a series of in vitro tests: calcium phosphate (CaP) growth in simulated body fluid (SBF), serum protein adsorption, and a morphology and attachment study of human osteoblast-like SaOS-2 cells. The graft copolymers were prepared by means of gamma irradiation induced grafting and displayed various surface morphologies and wettabilities depending on the grafting conditions used. Unmodified ePTFE did not induce nucleation of Cal? minerals, whereas all the grafted membranes revealed the growth of Cal? minerals after 7 days immersion in SBF. The sample with lowest surface grafting yield (24% coverage), a smooth graft morphology and relatively high hydrophobicity (theta(adv) = 120 degrees, theta(rec) = 80 degrees) showed carbonated hydroxyapatite growth covering the surface. On the other hand, the samples with high surface grafting yield (76% and 100%), a globular graft morphology and hydrophilic surfaces (theta(adv) = 60 degrees and 80 degrees, theta(rec) = 25 degrees and 15 degrees, respectively) exhibited irregular growth of non-apatitic Cap minerals. Irreversibly adsorbed protein measured after a 1 h immersion in serum solution was quantified by the amount of nitrogen on the surface using XPS, as well as by weight increase. All grafted membranes adsorbed 3-6 times more protein than the unmodified membrane. The sample with the highest surface coverage adsorbed the most protein. Osteoblast-like SaOS-2 cells cultured for 3 h revealed significantly higher levels of cell attachment on all grafted membranes compared to unmodified ePTFE. Although the morphology of the cells was heterogeneous, in general, the higher grafted surfaces showed a much better cell morphology than both the low surface-grafted and the control unmodified sample. The suite of in vitro tests confirms that a judicious choice of grafted monomer such as the phosphate-containing methacrylate monomer (MOEP) significantly improves the bioactivity of ePTFE in vitro. (c) 2005 Elsevier Ltd. All rights reserved.

Histopathology of oligofructose-induced acute laminitis in heifers

Histopathology of the dermo-epidermal junction in the lamellar region of front claws was examined in 6 dairy heifers given an alimentary oligofructose overload and compared with sections from a control group of 6 heifers. Four of the 6 heifers administered oligofructose developed clinical signs of acute laminitis before they were euthanized. Postmortem samples from front claws were processed for histology. Eleven histopathologic characteristics were selected from the existing literature and used in a blinded evaluation of sections. In total, 104 front claw samples, including 8 samples from 2 cows having spontaneously occurring acute laminitis, were evaluated histologically using hematoxylin and eosin as well as periodic acid-Schiff staining. The major morphological features associated with oligofructose-induced acute clinical laminitis were stretching of lamellae, dermal edema, hemorrhage, changes in basal cell morphology, presence of white blood cells in dermis, and signs of basement membrane detachment. Changes at the lamellar junction of claw tissue affected by oligofructose-induced clinical laminitis resembled tissue from the 2 cows suffering from spontaneous acute clinical laminitis, and generally were consistent with existing descriptions of laminitis histopathology. Important exceptions to existing descriptions in the literature were stretching of lamellae and basement membrane changes. Not previously described, we considered these early signs of acute laminitis. In conclusion, this study documents that oligofructose-induced clinical laminitis is associated with histopathological changes at the lamellar interface. A weakened dermo-epidermal junction is a possible intermediate stage in the pathophysiology of bovine sole ulceration at the typical site.

Role of growth hormone receptor signaling in osteogenesis from murine bone marrow progenitor cells

Growth hormone (GH) regulates many of the factors responsible for controlling the development of bone marrow progenitor cells (BMPCs). The aim of this study was to elucidate the role of GH in osteogenic differentiation of BMPCs using GH receptor null mice (GHRKO). BMPCs from GHRKO and their wild-type (WT) littermates were quantified by flow cytometry and their osteogenic differentiation in vitro was determined by cell morphology, real-time RT-PCR, and biochemical analyses. We found that freshly harvested GHRKO marrow contains 3% CD34 (hernatopoietic lineage), 43.5% CD45 (monocyte/macrophage lineage), and 2.5% CD106 positive (CFU-F/BMPC) cells compared to 11.2%, 45%, and 3.4% positive cells for (WT) marrow cells, respectively. When cultured for 14 days under conditions suitable for CFU-F expansion, GHRKO marrow cells lost CD34 positivity, and were markedly reduced for CD45, but 3- to 4-fold higher for CD106. While WT marrow cells also lost CD34 expression, they maintained CD45 and increased CD106 levels by 16-fold. When BMPCs from GHRKO mice were cultured under osteogenic conditions, they failed to elongate, in contrast to WT cells. Furthermore, GHRKO cultures expressed less alkaline phosphatase, contained less mineralized calcium, and displayed lower osteocalcin expression than WT cells. However, GHRKO cells displayed similar or higher expression of cbfa-1, collagen 1, and osteopontin mRNA compared to WT. In conclusion, we show that GH has an effect on the proportions of hematopoietic and mesenchymal progenitor cells in the bone marrow, and that GH is essential for both the induction and later progression of osteogenesis. (c) 2005 Elsevier Inc. All rights reserved.

Overlap between neurodegenerative disorders

Neurodegenerative disorders are characterized by the formation of distinct pathological changes in the brain, including extracellular protein deposits, cellular inclusions, and changes in cell morphology. Since the earliest published descriptions of these disorders, diagnosis has been based on clinicopathological features, namely, the coexistence of a specific clinical profile together with the presence or absence of particular types of lesion. In addition, the molecular profile of lesions has become an increasingly important feature both in the diagnosis of existing disorders and in the description of new disease entities. Recent studies, however, have reported considerable overlap between the clinicopathological features of many disorders leading to difficulties in the diagnosis of individual cases and to calls for a new classification of neurodegenerative disease. This article discusses: (i) the nature and degree of the overlap between different neurodegenerative disorders and includes a discussion of Alzheimer's disease, dementia with Lewy bodies, the fronto-temporal dementias, and prion disease; (ii) the factors that contribute to disease overlap, including historical factors, the presence of disease heterogeneity, age-related changes, the problem of apolipoprotein genotype, and the co-occurrence of common diseases; and (iii) whether the current nosological status of disorders should be reconsidered.

Are pathological lesions in neurodegenerative disorders the cause or the effect of the degeneration?

Pathological lesions in the form of extracellular protein deposits, intracellular inclusions and changes in cell morphology occur in the brain in the majority of neurodegenerative disorders. Studies of the presence, distribution, and molecular determinants of these lesions are often used to define individual disorders and to establish the mechanisms of lesion pathogenesis. In most disorders, however, the relationship between the appearance of a lesion and the underlying disease process is unclear. Two hypotheses are proposed which could explain this relationship: (i) lesions are the direct cause of the observed neurodegeneration ('causal' hypothesis); and (ii) lesions are a reaction to neurodegeneration ('reaction' hypothesis). These hypotheses are considered in relation to studies of the morphology and molecular determinants of lesions, the effects of gene mutations, degeneration induced by head injury, the effects of experimentally induced brain lesions, transgenic studies and the degeneration of anatomical pathways. The balance of evidence suggests that in many disorders, the appearance of the pathological lesions is a reaction to degenerative processes rather than being their cause. Such a conclusion has implications both for the classification of neurodegenerative disorders and for studies of disease pathogenesis.

Improper organization of the actin cytoskeleton affects protein synthesis at initiation

Although the actin cytoskeleton and the translation machinery are considered to be separate cellular complexes, growing evidence supports overlapping regulation of the two systems. Because of its interaction with actin, the eukaryotic translation elongation factor 1A (eEF1A) is proposed to be a regulator or link between these processes. Using a genetic approach with the yeast Saccharomyces cerevisiae, specific regions of eEF1A responsible for actin interactions and bundling were identified. Five new mutations were identified along one face of eEF1A. Dramatic changes in cell growth, cell morphology, and actin cable and patch formation as well as a unique effect on total translation in strains expressing the F308L or S405P eEF1A mutant form were observed. The translation effects do not correlate with reduced translation elongation but instead include an initiation defect. Biochemical analysis of the eEF1A mutant forms demonstrated reduced actin-bundling activity in vitro. Reduced total translation and/or the accumulation of 80S ribosomes in strains with either a mutation or a null allele of genes encoding actin itself or actin-regulating proteins Tpm1p, Mdm20p, and Bnirp/Bni1p was observed. Our data demonstrate that eEF1A, other actin binding proteins, and actin mutants affect translation initiation through the actin cytoskeleton.

What does the study of the spatial patterns of pathological lesions tell us about the pathogenesis of neurodegenerative disorders?

Discrete pathological lesions, which include extracellular protein deposits, intracellular inclusions and changes in cell morphology, occur in the brain in the majority of neurodegenerative disorders. These lesions are not randomly distributed in the brain but exhibit a spatial pattern, that is, a departure from randomness towards regularity or clustering. The spatial pattern of a lesion may reflect pathological processes affecting particular neuroanatomical structures and, therefore, studies of spatial pattern may help to elucidate the pathogenesis of a lesion and of the disorders themselves. The present article reviews first, the statistical methods used to detect spatial patterns and second, the types of spatial patterns exhibited by pathological lesions in a variety of disorders which include Alzheimer's disease, Down syndrome, dementia with Lewy bodies, Creutzfeldt-Jakob disease, Pick's disease and corticobasal degeneration. These studies suggest that despite the morphological and molecular diversity of brain lesions, they often exhibit a common type of spatial pattern (i.e. aggregation into clusters that are regularly distributed in the tissue). The pathogenic implications of spatial pattern analysis are discussed with reference to the individual disorders and to studies of neurodegeneration as a whole.

Tethering a laminin peptide to a crosslinked collagen scaffold for biofunctionality

Cell adhesion peptide regulates various cellular functions like proliferation, attachment, and spreading. The cellular response to laminin peptide (PPFLMLLKGSTR), a motif of laminin-5 alpha3 chain, tethered to type I collagen, crosslinked using microbial transglutaminase (mTGase) was investigated. mTGase is an enzyme that initiates crosslinking by reacting with the glutamine and lysine residues on the collagen fibers stabilizing the molecular structure. In this study that tethering of the laminin peptide in a mTGase crosslinked collagen scaffold enhanced cell proliferation and attachment. Laminin peptide tethered crosslinked scaffold showed unaltered cell morphology of 3T3 fibroblasts when compared with collagen and crosslinked scaffold. The triple helical structure of collagen remained unaltered by the addition of laminin peptide. In addition a dose-dependent affinity of the laminin peptide towards collagen was seen. The degree of crosslinking was measured by amino acid analysis, differential scanning calorimeter and fourier transform infrared spectroscopy. Increased crosslinking was observed in mTGase crosslinked group. mTGase crosslinking showed higher shrinkage temperature. There was alteration in the fibrillar architecture due to the crosslinking activity of mTGase. Hence, the use of enzyme-mediated linking shows promise in tethering cell adhesive peptides through biodegradable scaffolds.

The postantibiotic effect of the carbapenem antibiotics on gram-negative bacteria

Postantibiotic effect (PAE) describes the suppression of microbial growth occurring after a short exposure to an antimicrobial agent. PAE appears to be a property of the majority of antimicrobial agents and is demonstrated by a wide variety of microorganisms. At present, carbapenems and penems are the only members of the -lactam group of antimicrobial agents that exhibit a significant PAE on Gram-negative bacilli. A standardised method was developed to evaluate the in vitro PAE of three carbapenems; imipenem, meropenem and biapenem on Gram-negative bacteria under reproducible laboratory conditions that partially mimicked those occurring in vivo. The effects on carbapenem PAE of the method of antimicrobial removal, concentration, exposure duration, inoculum size, inoculum growth phase, multiple exposures and pooled human serum were determined. Additionally, the reproducibility, susceptibility prior to and after PAE determination and inter-strain variation of carbapenem PAE were evaluated. The method developed determined PAE by utilising viable counts and demonstrated carbapenem PAE to be reproducible, constant over successive exposures, dependent on genera, concentration, duration of exposure, inoculum size and growth phase. In addition, carbapenem PAE was not significantly effected either by agitation, the antimicrobial removal method or the viable count diluent. At present, the mechanism underlying PAE is undetermined. It is thought to be due to either the prolonged persistence of the antimicrobial at the cellular site of action or the true recovery period from non-lethal damage. Increasing the L-lysine concentration and salinity at recovery decreased and increased the carbapenem and imipenem PAE of Pseudomonas aeruginosa, respectively. In addition, no apparent change was observed in the production of virulence factors by P.aeruginosa in PAE phase. However, alterations in cell morphology were observed throughout PAE phase, and the reappearance of normal cell morphology corresponded to the duration of PAE determined by viable count. Thus, the recovery of the penicillin binding protein target enzymes appears to be the mechanism behind carbapenem PAE in P. aeruginosa.

Insulin gene therapy for the treatment of diabetes mellitus

Currently available treatments for insulin-dependent diabetes mellitus are often inadequate in terms of both efficacy and patient compliance. Gene therapy offers the possibility of a novel and improved method by which exogenous insulin can be delivered to a patient. This was approached in the present study by constructing a novel insulin-secreting cell line. For the purposes of this work immortalized cell lines were used. Fibroblasts and pituitary cells were transfected with the human preproisinulin gene to create stable lines of proinsulin- and insulin-secreting cells. The effect of known β-cell secretagogues on these cells were investigated, and found mostly to have no stimulatory effect, although IBMX, arginine and ZnSO4 each increased the rate of secretion. Cyclosporin (CyA) is currently the immunosuppresant of choice for transplant recipients; the effect of this treatment on endogenous β-cell function was assessed both in vivo and in vitro. Therapeutic doses of CyA were found to reduce plasma insulin concentrations and to impair glucose tolerance. The effect of immunoisolation on insulin release by HIT T15 cells was also investigated. The presence of an alginate membrane was found to severely impair insulin release. For the first implantation of the insulin-secreting cells, the animal model selected was the athymic nude mouse. This animal is immunoincompetent, and hence the use of an immunosuppressive regimen is circumvented. Graft function was assessed by measurement of plasma human C peptide concentrations, using a highly specific assay. Intraperitoneal implantation of genetically manipulated insulin-secreting pituitary cells into nude mice subsequently treated with a large dose of streptozotocin (STZ) resulted in a significantly delayed onset of hyperglycaemia when compared to control animals. Consumption of a ZnSO4 solution was shown to increase human C peptide release by the implant. Ensuing studies in nude mice examined the efficacy of different implantation sites, and included histochemical examination of the tumours. Aldehyde fuchsin staining and immunocytochemical processing demonstrated the presence of insulin containing cells within the excised tissue. Following initial investigations in nude mice, implantation studies were performed in CyA-immunosuppressed normal and STZ-diabetic mice. Graft function was found to be less efficacious, possibly due to the subcutaneous implantation site, or to the immunosuppresive regimen. Histochemical and transmission electron microscopic analysis of the tumour-like cell clusters found at autopsy revealed necrosis of cells at the core, but essentially normal cell morphology, with dense secretory granules in peripheral cells. The thesis provides evidence that gene therapy offers a feasibly new approach to insulin delivery.

Amino acid, peptide and drug transport across monolayers of human intestinal (CAC0-2) cells in vitro

The properties of Caco-2 monolayers were compared on aluminium oxide and nitrocellulose permeable-supports. On nitrocellulose, Caco-2 cells displayed a higher rate of taurocholic acid transport than those cultured on aluminium oxide inserts. In addition, Caco-2 cells grown on these two inserts were not comparable with respect to cell morphology, cell numbers and transepithelial electrical resistance. The low adsorption potential of the aluminium oxide inserts, particularly for high molecular weight or lipophilic ligands, offers a distinct advantage over nitrocellulose inserts for drug transport studies. The carrier-mediated uptake and transport of the imino acid (L-proline) and the acidic amino acids (L-aspartate and L-glutamate) have been studied. At pH7.4, L-proline uptake is mediated via an A-system carrier. Elevated uptake and transport under acidic conditions occurs by activation of a distinct carrier population. Acidic amino acid transport is mediated via a X-AG system. The flux of baclofen, CGP40116 andCGP40117 across Caco-2 monolayers was described by passive transport. The transport of three peptides, thyrotrophin-releasing hormone, SQ29852 and cyclosporin were investigated. Thyrotrophin-releasing hormone transport acrossCaco-2 monolayers was characterised by a minor saturable (carrier-mediated,approximately 25%) pathway, superimposed onto a major non-saturable (diffusional)pathway. SQ29852 uptake into Caco-2 monolayers is described by a major saturable mechanism (Km = 0.91 mM) superimposed onto a minor passive component.However, the initial-rate of SQ29852 transport is consistent with a passive transepithelial transport mechanism. These data highlight the possibility that itsbasolateral efflux is severely retarded such that the passive paracellular transportdictates the overall transepithelial transport characteristics. In addition, modelsuitable for investigating the transepithelial transport of cyclosporin A has been developed. A modification of the conventional Caco-2 model has been developed which has a calcium-free Ap donor-solution and a Bl receiver-solution containing the minimumcalcium concentration required to maintain monolayer integrity (100 μM). The influence of calcium and magnesium on the absorption of [14C]pamidronate was evaluated by comparing its transport across the conventional and minimum calciumCaco-2 models. Ap calcium and magnesium ions retard the Ap-to-Bl flux of pamidronate across Caco-2 monolayers. The effect of self-emulsifying oleic acid-Tween 80 formulations on Caco-2monolayer integrity has been investigated. Oleic acid-Tween 80 (1 0:1) formulations produced a dose-dependent disruption of Caco-2 monolayer integrity. This disruption was related to the oleic acid content of the formulation.

Characterisation of electrospun polystyrene scaffolds for three-dimensional in vitro biological studies

The purpose of this study was to produce a well-characterised electrospun polystyrene scaffold which could be used routinely for three-dimensional (3D) cell culture experimentation. A linear relationship (p<0.01p<0.01) between three principal process variables (applied voltage, working distance and polymer concentration) and fibre diameter was reliably established enabling a mathematical model to be developed to standardise the electrospinning process. Surface chemistry and bulk architecture were manipulated to increase wetting and handling characteristics, respectively. X-ray photoelectron spectroscopy (XPS) confirmed the presence of oxygen-containing groups after argon plasma treatment, resulting in a similar surface chemistry to treated tissue culture plastic. The bulk architecture of the scaffolds was characterised by scanning electron microscopy (SEM) to assess the alignment of both random and aligned electrospun fibres, which were calculated to be 0.15 and 0.66, respectively. This compared to 0.51 for collagen fibres associated with native tissue. Tensile strength and strain of approximately of 0.15 MPa and 2.5%, respectively, allowed the scaffolds to be routinely handled for tissue culture purposes. The efficiency of attachment of smooth muscle cells to electrospun scaffolds was assessed using a modified 3-[4,5-dimethyl(thiazol-2yl)-3,5-diphery] tetrazolium bromide assay and cell morphology was assessed by phalloidin-FITC staining of F-actin. Argon plasma treatment of electrospun polystyrene scaffold resulted in significantly increased cell attachment (p<0.05p<0.05). The alignment factors of the actin filaments were 0.19 and 0.74 for the random and aligned scaffold respectively, compared to 0.51 for the native tissue. The data suggests that electrospinning of polystyrene generates 3D scaffolds which complement polystyrene used in 2D cell culture systems.

Understanding the link between transglutaminase and the induction of fibrosis in cystic fibrosis (CF)

The emerging role of the multifunctional enzyme, Transglutaminase 2 (TG2) in Cystic Fibrosis (CF) has been linked to its increased expression and intracellular transamidating activity. However, a full understanding of the molecular mechanisms involved still remains unclear despite numerous studies that have attempted to delineate this process. These mechanisms include the NFκB and TGFβ1 pathway amongst others. This study reveals for the first time that the development of fibrosis in CF is due to a TG2-driven epithelial to mesenchymal transition (EMT) via a mechanism involving the activation of the pro-fibrotic cytokine TGFβ1. Using a human ΔF508/W1282X CFTR CF mutant bronchial cell (IB3-1), its CFTR corrected “add-back” cell (C38) as well as a primary human bronchial epithelial cell (HBEC), elevated TG2 levels in the CFTR mutant IB3 cell were shown to activate latent TGFβ1 leading to increased levels found in the culture medium. This activation process was blocked by the presence of cell-permeable and impermeable TG2 inhibitors while inhibition of TGFβ1 receptors blocked TG2 expression. This demonstrates the direct link between TG2 and TGFβ1 in CF. The presence of active cell surface TG2 correlated with an increase in the expression of EMT markers, associated with the CF mutant cells, which could be blocked by the presence of TG2 inhibitors. This was mimicked using the “addback” C38 cell and the primary human bronchial epithelial cell, HBEC, where an increase in TG2 expression and activity in the presence of TGFβ1 concurred with a change in cell morphology and an elevation in EMT marker expression. Conversely, a knockdown of TG2 in the CF mutant IB3 cells illustrated that an inhibition of TG2 blocks the increase in EMT marker expression as well as causing an increase in TEER measurement. This together with an increase in the migration profile of the CF mutant IB3 cell against the “add-back” C38 cell suggests that TG2 drives a mesenchymal phenotype in CF. The involvement of TG2 activated TGFβ1 in CF was further demonstrated with an elevation/inhibition of p- SMAD 2 and 3 activation in the presence of TGFβ1/TG2 cell-permeable/impermeable inhibitors respectively. The use of a comparative airway cell model where bronchial epithelial cells were cultured at the air liquid interface (ALI) confirmed the observations in submerged culture depicting the robustness of the model and reiterated the importance of TG2 in CF. Using a CFTR corrector combined with TG2 inhibitors, this study showed that the correction and stabilisation of the ΔF508 CFTR mutation in the mutant cell forged an increase in matured CFTR copies trafficking to the apical surface by circumventing proteosomal degradation. Thus the results presented here suggests that TG2 expression is elevated in the CFTR mutant bronchial cell via a TGFβ1 driven positive feedback cycle whereby activation of latent TGFβ1 by TG2 leads in turn to an elevation in its own expression by TGFβ1. This vicious cycle then drives EMT in CF ultimately leading to lung remodelling and fibrosis. Importantly, TG2 inhibition blocks TGFβ1 activation leading to an inhibition of EMT and further blocks the emerging fibrosis, thus stabilizing and supporting the maturation, trafficking and conductance of CFTR channels at the apical surface.