Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

Effects of different nutritional strategies on intestinal inflammation in pigs

Intestinal health is essential for the health of the body since the gastro-intestinal mucosa is the main site of interaction with the external environment, as well as the major area colonized by the microbiota. Intestinal health relies on proper barrier function, epithelial integrity and related mechanisms of protection (mucous layer, tight junctions, immune and inflammatory system). In pigs, during the weaning transition, intestinal inflammation and barrier integrity play a crucial role in regulating intestinal health and, consequently, pig’s health, growth and productivity. The aim of the project was to assess the impact of different nutritional strategies on the intestinal health of weaning piglets with reference to the inflammatory status and epithelial integrity. Therefore, in vivo trials were conducted to test the in-feed supplementation with zinc, tributyrin, or organic acids and nature-identical compounds (NIC) to weaning piglets. All the dietary interventions positively impacted the intestinal inflammatory status and, as a consequence, improved epithelial integrity by modulating tight junctions proteins (zinc or tributyrin) or by enhancing barrier properties measured with Ussing chambers (organic acids and NIC). These findings highlight that intestinal inflammation and barrier function are strictly linked, and that the control of inflammation is essential for adequate barrier function. In addition, in zinc trial and organic acids and NIC trial, better intestinal health could successfully result in better growth performance, as aimed for pig production improvement. To conclude, this work shows that dietary supplementation with bio-active substances such as zinc, tributyrin or organic acids and NIC may improve intestinal health of weaning piglets modulating intestinal inflammatory stress and barrier integrity and allowing better piglet’s health, growth and productivity.

Caveolin-1 opens endothelial cell junctions by targeting catenins

A fundamental phenomenon in inflammation is the loss of endothelial barrier function, in which the opening of endothelial cell junctions plays a central role. However, the molecular mechanisms that ultimately open the cell junctions are largely unknown.

Eosinophils in the gastrointestinal tract: friends or foes?

Eosinophils play an important role in the mucosal immune system of the gastrointestinal tract under resting and under inflammatory conditions. Under steady-state conditions, the mucosa of the digestive tract is the only organ harboring a substantial number of eosinophils, which, if need be, get activated and exert several effector and immunoregulatory functions. The precise function of these late-phase inflammatory cells is not yet completely understood. Nevertheless, it has recently been demonstrated that lipopolysaccharides from gram-negative bacteria activate eosinophils to rapidly release mitochondrial DNA in the extracellular space. Released mitochondrial DNA and eosinophil granule proteins form extracellular structures able to bind and inactivate bacteria. These findings suggest a novel mechanism of eosinophil-mediated innate immune responses that might be important in maintaining the intestinal barrier function. Moreover, eosinophils also play a crucial role in several inflammatory conditions, such as intestinal infections, immune-mediated inflammations and hypersensitivity reactions. Under chronic inflammatory conditions, the ability of the eosinophils to induce repair can lead to pathological sequelae in the tissue, such as esophageal remodeling in eosinophilic esophagitis. It is established that the uncontrolled eosinophilic inflammation induces fibrosis, esophageal wall thickening and strictures leading to damage that results in a loss of esophageal function. One potential mechanism of this remodeling is so-called 'epithelial mesenchymal transition', which is triggered by eosinophils and is potentially reversible under successful anti-eosinophil treatment. Therefore, eosinophils may act either as friends or as foes, depending on the microenvironment.

Intestinal bacterial translocation in rats with cirrhosis is related to compromised Paneth cell antimicrobial host defense

Liver cirrhosis is associated with bacterial translocation (BT) and endotoxemia. Most translocating bacteria belong to the common intestinal microbiota, suggesting a breakdown of intestinal barrier function. We hypothesized that diminished mucosal antimicrobial host defense could predispose to BT. Two rodent models of portal hypertension with increased BT were used, CCl(4)-induced ascitic cirrhosis and 2-day portal vein-ligated (PVL) animals. BT was assessed by standard microbiological techniques on mesenteric lymph nodes. Total RNA was isolated systematically throughout the intestinal tract, and expression of Paneth cell α-cryptdins and β-defensins was determined by real-time quantitative polymerase chain reaction (qPCR). To determine functional consequences, mucosal antimicrobial activity was assessed with a fluorescence-activated cell sorting assay. BT was detectable in 40% of rats with cirrhosis. Compared with the group without BT, these animals exhibited diminished intestinal Paneth cell α-cryptdin 5 and 7 expression. In contrast, PVL was associated with BT in all animals but did not affect antimicrobial peptides. The decrease in Paneth cell antimicrobials was most pronounced in the ileum and the coecum. Other antimicrobials showed no changes or even an induction in the case of BT at different sites. Antimicrobial activity toward different commensal strains was reduced, especially in the distal ileum and the cecum in experimental cirrhosis with BT (excluding PVL). Conclusion: Compromised Paneth cell antimicrobial host defense seems to predispose to BT in experimental cirrhosis. Understanding this liver-gut axis including the underlying mechanisms could help us to find new treatment avenues.

Mutations in the tight-junction gene claudin 19 (CLDN19) are associated with renal magnesium wasting, renal failure, and severe ocular involvement

Claudins are major components of tight junctions and contribute to the epithelial-barrier function by restricting free diffusion of solutes through the paracellular pathway. We have mapped a new locus for recessive renal magnesium loss on chromosome 1p34.2 and have identified mutations in CLDN19, a member of the claudin multigene family, in patients affected by hypomagnesemia, renal failure, and severe ocular abnormalities. CLDN19 encodes the tight-junction protein claudin-19, and we demonstrate high expression of CLDN19 in renal tubules and the retina. The identified mutations interfere severely with either cell-membrane trafficking or the assembly of the claudin-19 protein. The identification of CLDN19 mutations in patients with chronic renal failure and severe visual impairment supports the fundamental role of claudin-19 for normal renal tubular function and undisturbed organization and development of the retina.

Continuous thoracic epidural anesthesia improves gut mucosal microcirculation in rats with sepsis

Microcirculatory dysfunction contributes significantly to tissue hypoxia and multiple organ failure in sepsis. Ischemia of the gut and intestinal hypoxia are especially relevant for the evolution of sepsis because the mucosal barrier function may be impaired, leading to translocation of bacteria and toxins. Because sympathetic blockade enhances intestinal perfusion under physiologic conditions, we hypothesized that thoracic epidural anesthesia (TEA) may attenuate microcirculatory perturbations during sepsis. The present study was designed as a prospective and controlled laboratory experiment to assess the effects of continuous TEA on the mucosal microcirculation in a cecal ligation and perforation model of sepsis in rats. Anesthetized Sprague-Dawley rats underwent laparotomy and cecal ligation and perforation to induce sepsis. Subsequently, either bupivacaine 0.125% (n = 10) or isotonic sodium chloride solution (n = 9) was continuously infused via the thoracic epidural catheter for 24 h. In addition, a sham laparotomy was carried out in eight animals. Intravital videomicroscopy was then performed on six to ten villi of ileum mucosa. The capillary density was measured as areas encircled by perfused capillaries, that is, intercapillary areas. The TEA accomplished recruitment of microcirculatory units in the intestinal mucosa by decreasing total intercapillary areas (1,317 +/- 403 vs. 1,001 +/- 236 microm2) and continuously perfused intercapillary areas (1,937 +/- 512 vs. 1,311 +/- 678 microm2, each P < 0.05). Notably, TEA did not impair systemic hemodynamic variables beyond the changes caused by sepsis itself. Therefore, sympathetic blockade may represent a therapeutic option to treat impaired microcirculation in the gut mucosa resulting from sepsis. Additional studies are warranted to assess the microcirculatory effects of sympathetic blockade on other splanchnic organs in systemic inflammation.

The metalloprotease meprinbeta processes E-cadherin and weakens intercellular adhesion

BACKGROUND: Meprin (EC 3.4.24.18), an astacin-like metalloprotease, is expressed in the epithelium of the intestine and kidney tubules and has been related to cancer, but the mechanistic links are unknown. METHODOLOGY/PRINCIPAL FINDINGS: We used MDCK and Caco-2 cells stably transfected with meprin alpha and or meprin beta to establish models of renal and intestinal epithelial cells expressing this protease at physiological levels. In both models E-cadherin was cleaved, producing a cell-associated 97-kDa E-cadherin fragment, which was enhanced upon activation of the meprin zymogen and reduced in the presence of a meprin inhibitor. The cleavage site was localized in the extracellular domain adjacent to the plasma membrane. In vitro assays with purified components showed that the 97-kDa fragment was specifically generated by meprin beta, but not by ADAM-10 or MMP-7. Concomitantly with E-cadherin cleavage and degradation of the E-cadherin cytoplasmic tail, the plaque proteins beta-catenin and plakoglobin were processed by an intracellular protease, whereas alpha-catenin, which does not bind directly to E-cadherin, remained intact. Using confocal microscopy, we observed a partial colocalization of meprin beta and E-cadherin at lateral membranes of incompletely polarized cells at preconfluent or early confluent stages. Meprin beta-expressing cells displayed a reduced strength of cell-cell contacts and a significantly lower tendency to form multicellular aggregates. CONCLUSIONS/SIGNIFICANCE: By identifying E-cadherin as a substrate for meprin beta in a cellular context, this study reveals a novel biological role of this protease in epithelial cells. Our results suggest a crucial role for meprin beta in the control of adhesiveness via cleavage of E-cadherin with potential implications in a wide range of biological processes including epithelial barrier function and cancer progression.

Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense

Although eosinophils are considered useful in defense mechanisms against parasites, their exact function in innate immunity remains unclear. The aim of this study is to better understand the role of eosinophils within the gastrointestinal immune system. We show here that lipopolysaccharide from Gram-negative bacteria activates interleukin-5 (IL-5)- or interferon-gamma-primed eosinophils to release mitochondrial DNA in a reactive oxygen species-dependent manner, but independent of eosinophil death. Notably, the process of DNA release occurs rapidly in a catapult-like manner--in less than one second. In the extracellular space, the mitochondrial DNA and the granule proteins form extracellular structures able to bind and kill bacteria both in vitro and under inflammatory conditions in vivo. Moreover, after cecal ligation and puncture, Il5-transgenic but not wild-type mice show intestinal eosinophil infiltration and extracellular DNA deposition in association with protection against microbial sepsis. These data suggest a previously undescribed mechanism of eosinophil-mediated innate immune responses that might be crucial for maintaining the intestinal barrier function after inflammation-associated epithelial cell damage, preventing the host from uncontrolled invasion of bacteria.

A novel cell compatible impingement system to study in vitro drug absorption from dry powder aerosol formulations

A modified Astra type multistage liquid impinger (MSLI) with integrated bronchial cell monolayers was used to study deposition and subsequent drug absorption on in vitro models of the human airway epithelial barrier. Inverted cell culture of Calu-3 cells on the bottom side of cell culture filter inserts was integrated into a compendial MSLI. Upside down cultivation did not impair the barrier function, morphology and viability of Calu-3 cells. Size selective deposition with subsequent absorption was studied for three different commercially available dry powder formulations of salbutamol sulphate and budesonide. After deposition without size separation the absorption rates from the aerosol formulations differed but correlated with the size of the carrier lactose particles. However, after deposition in the MSLI, simulating relevant impaction and causing the separation of small drug crystals from the carrier lactose, the absorption rates of the three formulations were identical, confirming the bioequivalence of the three formulations.

The channel-activating protease CAP1/Prss8 is required for placental labyrinth maturation

The serine protease CAP1/Prss8 is crucial for skin barrier function, lung alveolar fluid clearance and has been unveiled as diagnostic marker for specific cancer types. Here, we show that a constitutive knockout of CAP1/Prss8 leads to embryonic lethality. These embryos presented no specific defects, but it is during this period, and in particular at E13.5, that wildtype placentas show an increased expression of CAP1/Prss8, thus suggesting a placental defect in the knockout situation. The placentas of knockout embryos exhibited significantly reduced vascular development and incomplete cellular maturation. In contrary, epiblast-specific deletion of CAP1/Prss8 allowed development until birth. These CAP1/Prss8-deficient newborns presented abnormal epidermis, and died soon after birth due to impaired skin function. We thus conclude that a late placental insufficiency might be the primary cause of embryonic lethality in CAP1/Prss8 knockouts. This study highlights a novel and crucial role for CAP1/Prss8 in placental development and function.

PURINERGIC SIGNALING REGULATES FILOPODIA-INDUCED ZIPPERING

The molecular mechanisms that mediate endometrial cancer invasion and metastasis remain poorly understood. This is a significant clinical problem, as there is no definitive cure for metastatic disease. The purinergic pathway’s generation of adenosine and its activation of the adenosine receptor A2B (A2BR) induces cell-cell adhesion to promote barrier function. This barrier function is known to be important in maintaining homeostasis during hypoxia, trauma, and sepsis. Loss of this epithelial barrier function provides a considerable advantage for carcinoma progression, as loss of cell-cell adhesions supports proliferation, aberrant signaling, epithelial-to-mesenchymal transition, invasion, and metastasis. The present work provides strong evidence that CD73-generated adenosine actively promotes cell-cell adhesion in carcinoma cells by filopodia-induced zippering. Adenosine-generating ecto-enzyme, CD73, was down-regulated in moderately- and poorly-differentiated, invasive, and metastatic endometrial carcinomas. CD73 expression and enzyme activity in normal endometrium and endometrial carcinomas was significantly correlated to the epithelial phenotype. Barrier function in normal epithelial cells of the endometrium was dependent on stress-induced generation of adenosine by CD73 and adenosine’s activation of A2BR. This same mechanism inhibited endometrial carcinoma cell migration and invasion. Finally, adenosine’s activation of A2BR induced the formation of filopodia that promoted the re-forming of cell-cell adhesions in carcinoma cells. Overall, these studies identified purinergic pathway-induced filopodia to be a novel mechanism of adenosine’s barrier function and a mechanism that has to be avoided/down-regulated by endometrial carcinoma cells attempting to lose attachment with their neighboring cells. These results provide insight into the molecular mechanisms of endometrial cancer invasion. In addition, because loss of cell-cell adhesions has been closely linked to therapy resistance in cancer, these results provide a rational clinical strategy for the re-establishment of cell-cell adhesions to potentially increase therapeutic sensitivity. In contrast to other molecular mechanisms regulating cell-cell adhesions, the purinergic pathway is clinically druggable, with agonists and antagonists currently being tested in clinical trials of various diseases.

The long non-coding RNA NEAT1 is increased in IUGR placentas, leading to potential new hypotheses of IUGR origin/development.

INTRODUCTION Intrauterine Growth Restriction (IUGR) is a multifactorial disease defined by an inability of the fetus to reach its growth potential. IUGR not only increases the risk of neonatal mortality/morbidity, but also the risk of metabolic syndrome during adulthood. Certain placental proteins have been shown to be implicated in IUGR development, such as proteins from the GH/IGF axis and angiogenesis/apoptosis processes. METHODS Twelve patients with term IUGR pregnancy (birth weight < 10th percentile) and 12 CTRLs were included. mRNA was extracted from the fetal part of the placenta and submitted to a subtraction method (Clontech PCR-Select cDNA Subtraction). RESULTS One candidate gene identified was the long non-coding RNA NEAT1 (nuclear paraspeckle assembly transcript 1). NEAT1 is the core component of a subnuclear structure called paraspeckle. This structure is responsible for the retention of hyperedited mRNAs in the nucleus. Overall, NEAT1 mRNA expression was 4.14 (±1.16)-fold increased in IUGR vs. CTRL placentas (P = 0.009). NEAT1 was exclusively localized in the nuclei of the villous trophoblasts and was expressed in more nuclei and with greater intensity in IUGR placentas than in CTRLs. PSPC1, one of the three main proteins of the paraspeckle, co-localized with NEAT1 in the villous trophoblasts. The expression of NEAT1_2 mRNA, the long isoform of NEAT1, was only modestly increased in IUGR vs. CTRL placentas. DISCUSSION/CONCLUSION The increase in NEAT1 and its co-localization with PSPC1 suggests an increase in paraspeckles in IUGR villous trophoblasts. This could lead to an increased retention of important mRNAs in villous trophoblasts nuclei. Given that the villous trophoblasts are crucial for the barrier function of the placenta, this could in part explain placental dysfunction in idiopathic IUGR fetuses.

Effects of systematic patient education in skin care and protection in a hand eczema clinic.

BACKGROUND Hand eczema has a high impact on patients' quality of life. The treatment focuses on improving skin barrier function. OBJECTIVES To evaluate the effects and acceptance of a novel educational program for patients with hand eczema. METHODS Retrospectively, the records of 36 patients who attended the prevention program and follow-up visits were analyzed. Physician global assessment (PGA) scores, acceptance and behavioral changes were assessed. RESULTS In 67% of patients, an improvement of the hand eczema could be attributed to the effects of our educational program. The mean PGA score significantly decreased from 3 before education to 2.2 during follow-up. Behavioral changes in both skin care and protection were reported in 81 and 86%, respectively. CONCLUSIONs: Our educational program had a positive effect on clinical outcome as well as adherence to skin care and protection measures. Its integration in a hand eczema clinic was feasible and well accepted by the patients.

Systemic therapy for atopic dermatitis.

Systemic therapy for atopic dermatitis (AD) is indicated in patients with severe disease refractory to adequate topical treatment. Currently available drugs aim to decrease inflammation by suppressing and/or modulating immune responses and thus may indirectly improve skin barrier function, resulting in a decrease in clinical signs and symptoms in particular pruritus. Before considering systemic treatment, patient adherence to topical treatment including skin care has to be ensured. The selection of the drug depends on the disease severity, localization, complications, concomitant diseases, and age of the patient, but also on their availability and costs as well as the doctor's experience. Bearing in mind the potential risk of resistance, systemic therapy with antibiotics should be exclusively considered in clinically manifest infections such as in children. Here, we review recently published clinical trials and case reports on systemic therapy of pediatric and adult patients with AD to draw conclusions for clinical practice. Although AD is a common disease, controlled clinical studies investigating the efficacy of systemic drugs are scarce, except for cyclosporine, which has been approved for the therapy of severe AD.