1,720,975 research outputs found
Development of a cell motility characterization system for industrial biotechnological applications
No full textCell migration is a very complex mechanism linked to the inflammatory response as well as to several processes of cell biology and development.
The movement of cells in response to a chemokine gradient is the most popular form of interaction between cell environment and surface and specific surface receptor triggers an intracellular signaling pathway. This phenomenon is known as chemotaxis and is characteristic of neutrophil movements in response to the chemokine Interleuchin-8 (IL8) in a 3D space. Another paradigm of dynamic interaction between cells is the mechanism underlying the confluency of cells cultured in a Petri dish. This process is particularly studied in growing epithelial cell lines, and is influenced by still undefined external stimuli as well as by cell proliferation and cytoskeleton reorganization, that allow the contact and interaction between cells in 2D space. Chronic inflammation is an ideal condition for the study of cell migration and its de-regulation; understanding the mechanisms involved in cell motility and their putative modulation, is the first step in setting new appropriate models of study.
During PhD project, different cellular models have been used: IB3 cell lines belonging to patients with Cystic Fibrosis (caused by mutations of cystic fibrosis transmembrane regulator (CFTR)), and T84 cell lines, a popular model of study of Coeliac Disease, a common intolerance to proteins of wheat. Both disease are characterized by a pro-inflammatory milieu with high level of tissue Transglutaminase (TG2), a multi-functions enzyme with a defined role in several human pathologies.
Cystic Fibrosis is the prototype of diseases in which an uncontrolled production of IL8 leads to a dysregulated neutrophil recruitment. The IB3 cell lines have been used to understand a molecular mechanisms of enhanced IL8 production and their modulation. The results evidence i) the role of TG2 as a new pathogenic factor in Cystic Fibrosis; ii) the role of post-translational modifications of TG2 as a link between genetic defect of CFTR ad inflammation iii) the mechanisms of autophagy inhibition via ROS-TG2 axis, in epithelial airway cell line and mice model of CF. The consequence is a fine modulation of IL8 production. The system allows the design of a 3D model to study cell migration under IL8 diffusive flux in qualitative and quantitative ways.
Another important pathological system with chronic inflammation is celiac disease and the analysis of interactions of alimentary peptides with the frontline gut epithelium is a useful model of study. The epithelial cells are pivot in the innate immune activation in CD and cytoskeleton rearrangement is the earliest event in such a response to gliadin peptides. The data in T84 cell model show a gliadin peptide-driven pro-inflammatory environment as a consequence of its impaired lysosomal degradation. Moreover, alterations of motility and cytoskeletal reorganization emphasize the “toxic” effect of peptide. Therefore, CD offers an ideal opportunity to set up a 2D model of study of cell motility.
Understanding the mechanisms of migration and the identification of appropriate target of modulation of cell recruitment could allow wide industrial applications in biotechnology and will be successful to provide a useful tool to test potential therapeuthic molecules
How Effectively Can Oxidative Stress and Inflammation Be Reversed When CFTR Function Is Pharmacologically Improved?
No full textA critical challenge in the age of advanced modulator therapies is to understand and determine how effectively chronic oxidative stress and oxidative stress-induced inflammation can be reversed and physiological balance restored when CFTR function is pharmacologically improved. The triple therapy with elexacaftor–tezacaftor–ivacaftor (ETI) suggests that CFTR activity in individuals with at least one F508del mutation can be partially restored to about 50% of normal levels. Although incomplete, the partial recovery of CFTR function has been shown to drastically lower sputum pathogen content, enhance microbiome diversity, and lower inflammation markers within the first year of treatment in adolescents and adults with cystic fibrosis. However, despite these advancements, residual airway infection, oxidative stress and inflammation persist, with levels similar to other chronic lung conditions, like non-CF bronchiectasis. This persistence highlights the necessity for innovative antioxidant and anti-inflammatory treatments, in particular for individuals with advanced lung disease. To address this issue, emerging multi-omics technologies offer valuable tools to investigate the impact of modulator therapies on various molecular pathways. By analyzing changes in gene expression, epigenetic modifications, protein profiles and metabolic processes in airway-derived samples, it could be possible to uncover the mechanisms driving persistent oxidative stress and inflammation. These insights could pave the way for identifying new therapeutic targets to fully restore airway health and overall physiological balance
Effect of CFTR Modulators on Oxidative Stress and Autophagy in Non-CFTR-Expressing Cells
No full textThe triple combination therapy for cystic fibrosis (CF), including elexacaftor, tezacaftor and ivacaftor (ETI or Trikafta), has been shown to improve lung function and reduce pulmonary exacerbations, thereby enhancing the quality of life for most CF patients. Recent findings suggest that both the individual components and ETI may have potential off-target effects, highlighting the need to understand how these modulators impact cellular physiology, particularly in cells that do not express CF transmembrane conductance regulator (CFTR). We used HEK293 cells, as a cell model not expressing the CFTR protein, to evaluate the effect of ETI and each of its components on autophagic machinery and on the Rab5/7 components of the Rab pathway. We firstly demonstrate that the single modulators Teza and Iva, and the combinations ET and ETI, increased ROS production in the absence of their target while decreasing it in cells expressing the CFTR ∆F508del. This increase in cellular stress was followed by an increase in the total level of polyubiquitinated proteins as well as the p62 level and LC3II/LC3I ratio. Furthermore, we found that ETI had the opposite effect on Rabs by increasing Rab5 levels while decreasing Rab7. Interestingly, these changes were abolished by the expression of mutated CFTR. Overall, our data suggest that in the absence of their target, both the individual modulators and ETI increased ROS production and halted both autophagic flux and plasma membrane protein recycling
Theratyping of the Rare CFTR Genotype A559T in Rectal Organoids and Nasal Cells Reveals a Relevant Response to Elexacaftor (VX-445) and Tezacaftor (VX-661) Combination
Full text linkDespite the promising results of new CFTR targeting drugs designed for the recovery of F508del- and class III variants activity, none of them have been approved for individuals with selected rare mutations, because uncharacterized CFTR variants lack information associated with the ability of these compounds in recovering their molecular defects. Here we used both rectal organoids (colonoids) and primary nasal brushed cells (hNEC) derived from a CF patient homozygous for A559T (c.1675G>A) variant to evaluate the responsiveness of this pathogenic variant to available CFTR targeted drugs that include VX-770, VX-809, VX-661 and VX-661 combined with VX-445. A559T is a rare mutation, found in African-Americans people with CF (PwCF) with only 85 patients registered in the CFTR2 database. At present, there is no treatment approved by FDA (U.S. Food and Drug Administration) for this genotype. Short-circuit current (Isc) measurements indicate that A559T-CFTR presents a minimal function. The acute addition of VX-770 following CFTR activation by forskolin had no significant increment of baseline level of anion transport in both colonoids and nasal cells. However, the combined treatment, VX-661-VX-445, significantly increases the chloride secretion in A559T-colonoids monolayers and hNEC, reaching approximately 10% of WT-CFTR function. These results were confirmed by forskolin-induced swelling assay and by western blotting in rectal organoids. Overall, our data show a relevant response to VX-661-VX-445 in rectal organoids and hNEC with CFTR genotype A559T/A559T. This could provide a strong rationale for treating patients carrying this variant with VX-661-VX-445-VX-770 combination
Identification of an ultra-rare Alu insertion in the CFTR gene: Pitfalls and challenges in genetic test interpretation
Full text link: Cystic fibrosis (CF) is a life-limiting genetic disorder characterized by defective chloride ion transport due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Early detection through newborn screening programs significantly improves outcomes for individuals with CF by enabling timely intervention. Here, we report the identification of an Alu element insertion within the exon 15 of CFTR gene, initially overlooked in standard next-generation sequencing analyses. However, using traditional molecular techniques, based on polymerase chain reaction and Sanger sequencing, allowed the identification of the Alu element and the reporting of a correct diagnosis. Our analysis, based on bioinformatics tools and molecular techniques, revealed that the Alu element insertion severely affects the gene expression, splicing patterns, and structure of CFTR protein. In conclusion, this study emphasizes the importance of how the integration of human expertise and modern technologies represents a pivotal step forward in genomic medicine, ensuring the delivery of precision healthcare to individuals affected by genetic diseases
Towards a rational combination therapy of cystic fibrosis: How cystamine restores the stability of mutant CFTR
No full textCystic fibrosis (CF) is most frequently due to homozygous ΔF508-CFTR mutation. The ΔF508-CFTR protein is unstable in the plasma membrane (PM), even if it is rescued by pharmacological agents that prevent its intracellular retention and degradation. Restoring defective autophagy in CF airways by proteostasis regulators (such as cystamine and its reduced form, cysteamine) can rescue and stabilize ΔF508-CFTR at the PM, thus enabling the action of CFTR potentiators, which are pharmacological agents that stimulate the function of CFTR as an ion channel. The effects of cystamine extend for days (in vitro) and weeks (in vivo) beyond washout, suggesting that once peripheral proteostasis has been re-established, PM-resident ΔF508-CFTR sustains its own stability. We demonstrated that the pharmacological inhibition of wild-type CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette subfamily C, member 7)], in bronchial epithelial cells decreases the stability of the CFTR protein by inhibiting autophagy, elevating the abundance of SQSTM1/p62 and its interaction with CFTR at the PM, increasing the ubiqutination of CFTR, stimulating the lysosomal degradation of CFTR and avoiding its recycling. All these effects could be inhibited by cystamine. Moreover, CFTR-sufficient epithelia generate permissive conditions for incorporating ΔF508-CFTR into the PM and stabilizing it at this location. These results provide the rationale for a combination therapy of CF in which pretreatment with cystamine or cysteamine enables the later action of CFTR potentiators
Collective rotational motion of freely-expanding T84 epithelial cell colonies
Full text linkCoordinated rotational motion is an intriguing, yet still elusive mode of
collective cell migration, which is relevant in pathological and morphogenetic
processes. Most of the studies on this topic have been carried out on confined
epithelial cells. The driver of collective rotation in such conditions has not
been clearly elucidated, although it has been speculated that spatial
confinement can play an essential role in triggering cell rotation. Here, we
study the growth of epithelial cell colonies freely expanding (i.e., with no
physical constraints) on the surface of cell culture plates, a case which has
received scarce attention in the literature. We find that coordinated cell
rotation spontaneously occurs in cell clusters in the free growth regime, thus
implying that cell confinement is not necessary to elicit collective rotation
as previously suggested. The collective rotation was size and shape dependent:
a highly coordinated disk-like rotation was found in small cell clusters with a
round shape, while collective rotation was suppressed in large irregular cell
clusters generated by merging of different clusters in the course of their
growth. The angular motion was persistent in the same direction, although
clockwise and anticlockwise rotations were equally likely to occur among
different cell clusters. Radial cell velocity was low as compared to the
angular velocity. A clear difference in morphology was observed between cells
at the periphery and the ones in the core of the clusters, the former being
more elongated and spread out as compared to the latter. Overall, our results
provide the first quantitative and systematic evidence that coordinated cell
rotation does not require a spatial confinement and occurs spontaneously in
freely expanding epithelial cell colonies
Genistein antagonizes gliadin-induced CFTR malfunction in models of celiac disease
Full text linkIn celiac disease (CD), an intolerance to dietary gluten/gliadin, antigenic gliadin peptides trigger an HLA-DQ2/DQ8-restricted adaptive Th1 immune response. Epithelial stress, induced by other non-antigenic gliadin peptides, is required for gliadin to become fully immunogenic. We found that cystic-fibrosis-transmembrane-conductance-regulator (CFTR) acts as membrane receptor for gliadin-derived peptide P31-43, as it binds to CFTR and impairs its channel function. P31-43-induced CFTR malfunction generates epithelial stress and intestinal inflammation. Maintaining CFTR in an active open conformation by the CFTR potentiators VX-770 (Ivacaftor) or Vrx-532, prevents P31-43 binding to CFTR and controls gliadin-induced manifestations. Here, we evaluated the possibility that the over-the-counter nutraceutical genistein, known to potentiate CFTR function, would allow to control gliadin-induced alterations. We demonstrated that pre-treatment with genistein prevented P31-43-induced CFTR malfunction and an epithelial stress response in Caco-2 cells. These effects were abrogated when the CFTR gene was knocked out by CRISP/Cas9 technology, indicating that genistein protects intestinal epithelial cells by potentiating CFTR function. Notably, genistein protected gliadin-sensitive mice from intestinal CFTR malfunction and gliadin-induced inflammation as it prevented gliadin-induced IFN-γ production by celiac peripheral-blood-mononuclear-cells (PBMC) cultured ex-vivo in the presence of P31-43-challenged Caco-2 cells. Our results indicate that natural compounds capable to increase CFTR channel gating might be used for the treatment of CD
The immune and microbial homeostasis determines the Candida–mast cells cross-talk in celiac disease
No full textCeliac disease (CD) is an autoimmune enteropathy resulting from an interaction between diet, genome, and immunity. Although many patients respond to a gluten-free diet, in a substantive number of individuals, the intestinal injury persists. Thus, other factors might amplify the ongoing inflammation. Candida albicans is a commensal fungus that is well adapted to the intestinal life. However, specific conditions increase Candida pathogenicity. The hypothesis that Candida may be a trigger in CD has been proposed after the observation of similarity between a fungal wall component and two CD-related gliadin T-cell epitopes. However, despite being implicated in intestinal disorders, Candida may also protect against immune pathologies highlighting a more intriguing role in the gut. Herein, we postulated that a state of chronic inflammation associated with microbial dysbiosis and leaky gut are favorable conditions that promote C. albicans pathogenicity eventually contributing to CD pathology via a mast cells (MC)-IL-9 axis. However, the restoration of immune and microbial homeostasis promotes a beneficial C. albicans–MC cross-talk favoring the attenuation of CD pathology to alleviate CD pathology and symptoms
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