Marcella Cammarota

Divergence of gut permeability and mucosal immune gene expression in two gluten-associated conditions: celiac disease and gluten sensitivity

BackgroundCeliac disease (CD) is an autoimmune enteropathy triggered by the ingestion of gluten. Gluten-sensitive individuals (GS) cannot tolerate gluten and may develop gastrointestinal symptoms similar to those in CD, but the overall clinical picture is generally less severe and is not accompanied by the concurrence of tissue transglutaminase autoantibodies or autoimmune comorbidities. By studying and comparing mucosal expression of genes associated with intestinal barrier function, as well as innate and adaptive immunity in CD compared with GS, we sought to better understand the similarities and differences between these two gluten-associated disorders.MethodsCD, GS and healthy, gluten-tolerant individuals were enrolled in this study. Intestinal permeability was evaluated using a lactulose and mannitol probe, and mucosal biopsy specimens were collected to study the expression of genes involved in barrier function and immunity.ResultsUnlike CD, GS is not associated with increased intestinal permeability. In fact, this was significantly reduced in GS compared with controls (P = 0.0308), paralleled by significantly increased expression of claudin (CLDN) 4 (P = 0.0286). Relative to controls, adaptive immunity markers interleukin (IL)-6 (P = 0.0124) and IL-21 (P = 0.0572) were expressed at higher levels in CD but not in GS, while expression of the innate immunity marker Toll-like receptor (TLR) 2 was increased in GS but not in CD (P = 0.0295). Finally, expression of the T-regulatory cell marker FOXP3 was significantly reduced in GS relative to controls (P = 0.0325) and CD patients (P = 0.0293).ConclusionsThis study shows that the two gluten-associated disorders, CD and GS, are different clinical entities, and it contributes to the characterization of GS as a condition associated with prevalent gluten-induced activation of innate, rather than adaptive, immune responses in the absence of detectable changes in mucosal barrier function.

Enteroglial-derived S100B protein integrates bacteria-induced Toll-like receptor signalling in human enteric glial cells

Objective Enteric glial cells (EGC) have been suggested to participate in host–bacteria cross-talk, playing a protective role within the gut. The way EGC interact with microorganisms is still poorly understood. We aimed to evaluate whether: EGC participate in host–bacteria interaction; S100B and Toll-like receptor (TLR) signalling converge in a common pathway leading to nitric oxide (NO) production. Design Primary cultures of human EGC were exposed to pathogenic (enteroinvasive Escherichia coli; EIEC) and probiotic (Lactobacillus paracasei F19) bacteria. Cell activation was assessed by evaluating the expression of cFos and major histocompatibility complex (MHC) class II molecules. TLR expression in EGC was evaluated at both baseline and after exposure to bacteria by real-time PCR, fluorescence microscopy and western blot analysis. S100B expression and NO release from EGC, following exposure to bacteria, were measured in the presence or absence of specific TLR and S100B pathway inhibitors. Results EIEC activated EGC by inducing the expression of cFos and MHC II. EGC expressed TLR at baseline. Pathogens and probiotics differentially modulated TLR expression in EGC. Pathogens, but not probiotics, significantly induced S100B protein overexpression and NO release from EGC. Pretreatment with specific inhibitors of TLR and S100B pathways abolished bacterial-induced NO release from EGC. Conclusions Human EGC interact with bacteria and discriminate between pathogens and probiotics via a different TLR expression and NO production. In EGC, NO release is impaired in the presence of specific inhibitors of the TLR and S100B pathways, suggesting the presence of a novel common pathway involving both TLR stimulation and S100B protein upregulation.

The most widespread desmosomal cadherin, desmoglein 2, is a novel target of caspase 3-mediated apoptotic machinery

Apoptotic cells are known to regulate the ordered dismantling of intercellular contacts through caspase activity. Despite the important role of desmoglein (Dsg) 2 in epithelial cell–cell adhesion, the fate of this widespread desmosomal cadherin during apoptosis is yet poorly understood. Here, by means of pharmacological approaches, we investigated whether Dsg2 was targeted by caspases in HaCaT and HT‐29 cell lines undergoing staurosporine (STS)‐induced apoptosis. Results showed that STS induced a caspase‐dependent form of cell‐death in both keratinocytes (HaCaT) and enterocytes (HT‐29), that associated with progressive depletion of Dsg2 from cell lysates. The proteolytic processing of full‐length Dsg2 resulted in the appearance of a 70‐kDa fragment which was released into the cytosol. Consistently, immunofluorescence studies revealed that Dsg2 staining was abolished from cell surface whereas the cytoplasmic region of Dsg2 did localize intracellularly. Plakoglobin (Pg) also underwent cleavage and detached from Dsg2. Apoptotic changes paralleled with progressive loss of intercellular adhesion strength. All these biochemical, morphological, and functional changes were regulated by caspase 3. Indeed, in the presence of the caspase 3‐inhibitor z‐DEVD‐fmk, full‐length Dsg2 protein levels were preserved, whereas the amount of the 70‐kDa fragment was maintained on control levels. Furthermore, cells pretreated with z‐DEVD‐fmk retained the membrane labeling of Dsg2. Taken together, our data demonstrate that the apoptotic processing of Dsg2 is mediated by caspase 3 in epithelial cells. J. Cell. Biochem. 103: 598–606, 2008.

Effects of low concentrations of benzene on human lung cells in vitro.

Exposure to benzene causes health hazards to humans. The airway epithelium is a physical barrier to inhaled toxicants and particulates. This is an in vitro basic science study to evaluate the effects of benzene on lung cells without the inflammatory responses triggered by inhalation. Dose-response cytotoxicity was assessed using two cell lines: alveolar derived (A549) human epithelial adenocarcinoma and human lung (LL24) fibroblast. A549 cells were more resistant than LL24 fibroblast lung cells to benzene. LL24 cells demonstrated enhanced proliferation with diluted benzene solutions. Moreover, low concentrations of benzene enhanced telomerase activity in LL24 cells while no effects were observed in the adenocarcinoma cells. Proteolysis of lung matrix by matrix metalloproteinases (MMPs) is an early event observed in lung pathologies. Benzene increased MMP-2 and MMP-3 mRNA. Using the ratio (MMP-1+MMP-2+MMP-3)/(TIMP-1+TIMP-2), as an index of prodestructive activity, we observed a dose-dependent increase. The overall higher expression levels of MMPs in benzene treated cells did not appear to be controlled by TIMPs, which are negatively correlated. Comparing different cell lines, we demonstrated how crucial is the targets susceptibility. Our observations may represent early functional alterations that occur in the airways of exposed people.

Analysis of the signal transduction pathway of nickel-induced matrix metalloproteinase-2 expression in the human keratinocytes in vitro: preliminary findings

Background:  Nickel can induce cellular and nuclear damages responsible for chronic diseases, like allergic contact dermatitis (ACD). We previously showed that matrix metalloproteinase‐2 (MMP‐2) gene expression was induced by nickel in nontumorigenic human keratinocytes cell line (HaCat).

Hyaluronan hydrogels with a low degree of modification as scaffolds for cartilage engineering

In the field of cartilage engineering, continuing efforts have focused on fabricating scaffolds that favor maintenance of the chondrocytic phenotype and matrix formation, in addition to providing a permeable, hydrated, microporous structure and mechanical support. The potential of hyaluronan-based hydrogels has been well established, but the ideal matrix remains to be developed. This study describes the development of hyaluronan sponges-based scaffolds obtained by lysine methyl-ester crosslinking. The reaction conditions are optimized with minimal chemical modifications to obtain materials that closely resemble elements in physiological cellular environments. Three hydrogels with different amounts of crosslinkers were produced that show morphological, water-uptake, mechanical, and stability properties comparable or superior to those of currently available hyaluronan-scaffolds, but with significantly fewer hyaluronan modifications. Primary human chondrocytes cultured with the most promising hydrogel were viable and maintained lineage identity for 3 weeks. They also secreted cartilage-specific matrix proteins. These scaffolds represent promising candidates for cartilage engineering.

Epigenetic modulator UVI5008 inhibits MRSA by interfering with bacterial gyrase

The impact of multi-drug resistant bacterial strains on human health is reaching worrisome levels. Over 2 million people are infected by resistant bacteria, and more than 700,000 people die each year because of the continuous spread of resistant strains. The development of new antibiotics and the prudent use of existing ones to prolong their lifespan require a constant effort by drug industries and healthcare workers. The re-purposing of existing drugs for use as antimicrobial agents would streamline the development of new antibacterial strategies. As part of this effort, we screened a panel of drugs previously characterized to be epigenetic modulators/pro-apoptotic/differentiative drugs. We selected a few compounds that alter Gram-positive growth. Among these, UVI5008, a derivative of the natural compound psammaplin A (Psa_A), was identified. The interaction of Psa_A with the DNA gyrase enzyme has been shown, and here, we hypothesized and confirmed the gyrase-specific activity by biochemical assays. UVI5008 exhibited growth inhibition activity against Staphylococcus aureus via structural modification of the cell wall, which was observed by SEM electron microscopy. Based on our findings, we propose UVI5008 as an alternative antibacterial compound against methicillin-resistant (Met.R) S. aureus strains.

In vitro intestinal epithelium responses to titanium dioxide nanoparticles

Titanium dioxide (TiO2) is enclosed in many consumer products including pharmaceuticals, cosmetics, and foods. TiO2 (E171) is daily ingested as mixed nano- and submicron-sized particles since it is approved as a white colorant in Europe in a wide variety of food products, Noteworthy, the relevant risk assessment has never been satisfactorily concluded and growing alarms for human hazards deriving from TiO2 exposure are incrementally reported. The objective of the present study was to establish conceivable mechanisms by which nano-sized TiO2 particles affect physiological function of the intestinal epithelium layer. The well-established Caco-2 cell line differentiated for 21 days on permeable supports was used as a predictive model of the human intestinal mucosa to identify the biological response triggered by TiO2 particles. Exposure to 42 μg/mL TiO2 nanoparticles disrupted the tight junctions-permeability barrier with a prompt effect detectable after 4 h incubation time and wide effects on barrier integrity at 24 h. Transport and ultrastructural localization of TiO2 nanoparticles were determined by ICP-OES, TEM and ESI/EELS analysis, respectively. Nano-sized particles were efficiently internalized and preferentially entrapped by Caco-2 monolayers. Storage of TiO2 nanoparticles inside the cells affected enterocytes viability and triggered the production of pro-inflammatory cytokines, including TNF-α and IL-8. Taken together these data indicate that nano-sized TiO2 particles exert detrimental effects on the intestinal epithelium layer.

Macroporous alginate foams crosslinked with strontium for bone tissue engineering

Nowadays, the need of novel strategies to repair and regenerate bone defects in the field of biomedical applications has increased. Novel approaches include the design of natural bioactive scaffolds mimicking bone tissue. These bioactive scaffolds have to possess biophysical properties suitable to address biological response towards newly bone tissue formation. In particular, scaffold porosity and pore size play a pivotal role in cell migration, adhesion and proliferation, thus increasing cell-material surface interaction and osteogenic signals transmission. Here we propose the development of macroporous alginate foams (MAFs) with porous and well interconnected structure, useful to enhance growth and osteogenic differentiation of human Mesenchymal Stem Cells (hMSCs). Moreover, in this study we report a new method for MAFs fabrication based on the combination of internal gelation technique with gas foaming. Strontium was employed in combination with calcium as cross-linking agent for the alginate chains and as enhancer of the osteogenic differentiation. The influence of strontium ions on the gelation kinetics, physical properties and degradation in physiological medium of MAFs was investigated. Our results suggest that the combination of internal gelation technique with gas foaming followed by freeze-drying is an easy and straightforward procedure to prepare alginate foams with high porosity and interconnectivity, able to support cell infiltration. Finally, biological assays showed how scaffolds with high strontium content are able to support cell growth and differentiation in long times by promoting osteogenic marker expression.

Optical monitoring of cell migration processes in a 3D scaffold

Three-dimensional (3D) polymeric scaffolds are utilized in tissue engineering to provide biomechanical support for the seeded cells until they are organized into a functional tissue. Studies of cell migration are important for understanding a variety of physiological and pathological processes in tissue regeneration. Optical techniques can be employed for monitoring cell migration processes in 3D scaffolds. In particular, two-photon microscopy (TPM) can be used for monitoring these processes for living cells also at considerable depths. In the present work, the cell migration process of alive chondrocytes labelled with vital PKH67 in 3D collagen-based scaffold has been monitored at different times from the seeding process. The results show that TPM can evidence the presence of living cells in different spatial regions whose localizations depend on the location and time elapsed from the seeding.