[Show abstract][Hide abstract] ABSTRACT: To characterize longitudinally the inflammation and the gut microbiota dynamics in a mouse model of dextran sulfate sodium (DSS)-induced colitis.
In animal models, the most common method used to trigger colitis is based on the oral administration of the sulfated polysaccharides DSS. The murine DSS colitis model has been widely adopted to induce severe acute, chronic or semi-chronic colitis, and has been validated as an important model for the translation of mice data to human inflammatory bowel disease (IBD). However, it is now clear that models characterized by mild intestinal damage are more accurate for studying the effects of therapeutic agents. For this reason, we have developed a murine model of mild colitis to study longitudinally the inflammation and microbiota dynamics during the intestinal repair processes, and to obtain data suitable to support the recovery of gut microbiota-host homeostasis.
All plasma cytokines evaluated, except IL-17, began to increase (P < 0.05), after 7 d of DSS administration. IL-17 only began to increase 4 d after DSS withdrawal. IL-1β and IL-17 continue to increase during the recovery phase, even when clinical signs of colitis had disappeared. IL-6, IL-10 and IFN-γ reached their maxima 4 d after DSS withdrawal and decreased during the late recovery phase. TNFα reached a peak (a three- fold increase, P < 0.05), after which it slightly decreased, only to increase again close to the end of the recovery phase. DSS administration induced profound and rapid changes in the mice gut microbiota. After 3 d of DSS administration, we observed a major reduction in Bacteroidetes/Prevotella and a corresponding increase in Bacillaceae, with respect to control mice. In particular, Bacteroidetes/Prevotella decreased from a relative abundance of 59.42%-33.05%, while Bacillaceae showed a concomitant increase from 2.77% to 10.52%. Gut microbiota rapidly shifted toward a healthy profile during the recovery phase and returned normal 4 d after DSS withdrawal. Cyclooxygenase 2 expression started to increase 4 d after DSS withdrawal (P < 0.05), when dysbiosis had recovered, and continued to increase during the recovery phase. Taken together, these data indicated that a chronic phase of intestinal inflammation, characterized by the absence of dysbiosis, could be obtained in mice using a single DSS cycle.
Dysbiosis contributes to the local and systemic inflammation that occurs in the DSS model of colitis; however, chronic bowel inflammation is maintained even after recovery from dysbiosis.
World Journal of Gastroenterology 02/2014; 20(8):2051-61. · 2.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hemolytic uremic syndrome (HUS) caused by intestinal Shiga toxin-producing Escherichia coli infections is a worldwide health problem, as dramatically exemplified by the German outbreak occurred in summer 2011 and by a constant burden of cases in children. Shiga toxins (Stx) play a pivotal role in HUS by triggering endothelial damage in kidney and brain through globotriaosylceramide (Gb3Cer) receptor targeting. Moreover, Stx interact with human neutrophils, as experimentally demonstrated in vitro and as observed in patients with HUS. A neutrophil-protective role on endothelial damage (sequestration of circulating toxins) and a causative role in toxin delivery from the gut to the kidney (piggyback transport) have been suggested in different studies. However, the receptor that recognizes Stx in human neutrophils, which do not express Gb3Cer, has not been identified. In this study, by competition and functional experiments with appropriate agonists and antagonists (LPS, anti-TLR4 Abs, respectively), we have identified TLR4 as the receptor that specifically recognizes Stx1 and Stx2 in human neutrophils. Accordingly, these treatments displaced both toxin variants from neutrophils and, upon challenge with Stx1 or Stx2, neutrophils displayed the same pattern of cytokine expression as in response to LPS (assessed by quantitative RT-PCR, ELISA, or multiplexed Luminex-based immunoassays). Moreover, data were supported by adequate controls excluding any potential interference of contaminating LPS in Stx-binding and activation of neutrophils. The identification of the Stx-receptor on neutrophils provides additional elements to foster the understanding of the pathophysiology of HUS and could have an important effect on the development of therapeutic strategies.
The Journal of Immunology 09/2013; · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Colitis is the term used for chronic inflammatory bowel diseases at substantially increased risk of developing a form of colorectal cancer (CRC) known as colitis-associated cancer. In our study we synthesized core-shell polymeric micelles obtained by self-assembly of block copolymers for high efficiency delivery of anti-inflammatory and anti-cancer compounds to colonocytes and colon mucosa. We achieved an efficient intracellular delivery of these hydrophobic compounds (prednisone, retinoic acid and doxorubicin) to cultured colonocytes without cellular toxicity. The efficacy of retinoic acid and doxorubicin administration was significantly increased using these nanosized carriers. Moreover, these polymeric micelles have been shown to overcome the multidrug resistance efflux mechanism effectively delivering doxorubicin to multidrug-resistant colon cancer cells. These nanocarriers are also suitable for selective in vivo delivery of liphophilic drugs by enema administration to the inflamed colon tissue, specifically targeting the inflamed mucosa.
Nanomedicine: nanotechnology, biology, and medicine 12/2012; · 6.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: The vaginal microbiota of healthy women consists of a wide variety of anaerobic and aerobic bacterial genera and species dominated by the genus Lactobacillus. The activity of lactobacilli helps to maintain the natural healthy balance of the vaginal microbiota. This role is particularly important during pregnancy because vaginal dismicrobism is one of the most important mechanisms for preterm birth and perinatal complications. In the present study, we characterized the impact of a dietary supplementation with the probiotic VSL#3, a mixture of Lactobacillus, Bifidobacterium and Streptococcus strains, on the vaginal microbiota and immunological profiles of healthy women during late pregnancy. RESULTS: An association between the oral intake of the probiotic VSL#3 and changes in the composition of the vaginal microbiota of pregnant women was revealed by PCR-DGGE population profiling. Despite no significant changes were found in the amounts of the principal vaginal bacterial populations in women administered with VSL#3, qPCR results suggested a potential role of the probiotic product in counteracting the decrease of Bifidobacterium and the increase of Atopobium, that occurred in control women during late pregnancy. The modulation of the vaginal microbiota was associated with significant changes in some vaginal cytokines. In particular, the decrease of the anti-inflammatory cytokines IL-4 and IL-10 was observed only in control women but not in women supplemented with VSL#3. In addition, the probiotic consumption induced the decrease of the pro-inflammatory chemokine Eotaxin, suggesting a potential anti-inflammatory effect on the vaginal immunity. CONCLUSION: Dietary supplementation with the probiotic VSL#3 during the last trimester of pregnancy was associated to a modulation of the vaginal microbiota and cytokine secretion, with potential implications in preventing preterm birth.Trial RegistrationClinicalTrials.gov NCT01367470.
[Show abstract][Hide abstract] ABSTRACT: This study was aimed at determining the probiotic potential of a large number of autochthonous lactic acid bacteria isolated from fruit and vegetables. Survival under simulated gastric and intestinal conditions showed that 35% of the strains, mainly belonging to the species Lactobacillus plantarum maintained high cell densities. Selected strains did not affect the immune-mediation by Caco-2 cells. All strains stimulated all 27 immune-mediators by peripheral blood mononuclear cells (PBMC). A significant (P<0.05; P<0.01) increase of the major part of cytokines and growth factors was found. A few chemokines were stimulated. Immune-mediators with pro-inflammatory activity (IL-17, EOTAXIN and IFNγ) were significantly (P<0.01) stimulated by all strains, followed by IL-1b>IP-10>IL-6>MIP1α. Stimulation of IL-12, IL-2 and IL-7 was strain dependent. Only a few strains increased the synthesis of cytokines with anti-inflammatory activity. Six L. plantarum strains were further selected. Four were defined as the strongly adhesive strains (more than 40 bacteria adhering to one Caco-2 cell), and 2 as the adhesive strains (5-40 bacteria adhering to one Caco-2 cell). Five strains grew and acidified chemically defined medium with fructo-oligosaccharides (FOS) as the only carbon source. End-products of FOS fermentation were found. All strains inhibited enterohemorragic Escherichia coli K12 and Bacillus megaterium F6 isolated from human sources. The results of this study showed that some autochthonous lactic acid bacteria from raw fruit and vegetables have functional features to be considered as novel probiotic candidates.
[Show abstract][Hide abstract] ABSTRACT: PS-b-PMA block copolymers carrying terminal anionic groups have been studied. Their synthesis is achieved by mean of 3-(benzylsulfanyl thiocarbonylsulfanyl) propionic acid and 3-(1-phenylethylsulfanyl thiocarbonylsulfanyl) propanesulfonic acid sodium salt as RAFT agents. These block copolymers, when dispersed in methanol, display a pseudo-amphiphilic behaviour and provide micelles that can be suspended in water by osmosis. During the self-assembling process, the anionic terminations lead to the formation of nanosized monodispersed particles and confer stability to the micelles in aqueous media. The core of the micelles is loaded with Nile Red to achieve the intracellular delivery of a lipophilic substance; this has been demonstrated by Confocal Laser Scanning Microscopy. The particles, when loaded with Doxorubicin, are able to overcome the intrinsic resistance of LoVo-MDR cancer cells. A further stabilization is given to the micelles by covalent shell crosslinking provided by triethylene glycol diacrylate. The increased intracellular stability of the crosslinked micelles makes them stain, when loaded with Nile Red, the aqueous cytosol instead of the lipophilic cellular compartments. The unimers constituting the micelles have been covalently labelled with fluorescein to follow their fate once incorporated into the cells.
[Show abstract][Hide abstract] ABSTRACT: Aquatic organisms may suffer from exposure to high Cu concentrations, since this metal is widely used in feed supplementation, in pesticide formulation and as antifouling. Chronic exposure to Cu, even at sub-lethal doses, may strongly affect fish physiology. To date, several biomarkers have been used to detect Cu exposure in fish producing contrasting results. Therefore, we used a proteomic approach to clarify how Cu exposure may affect the serum proteome of gilthead sea bream (Sparus aurata), since serum could be considered a good source of early-biomarkers of Cu toxicosis. For this purpose we exposed juvenile gilthead sea bream to waterborne Cu (0.5 mg/L). Our results indicate that fish tightly regulate circulating Cu levels, which are not affected by metal exposure. This homeostatic control is mainly achieved by the liver, able to excrete high amounts of the metal via bile. Cu exposure caused differential expression of several serum proteins, 10 of which were identified by Mascot and BLAST search. All these proteins, with the exception of growth hormone receptor and γ-glutamyl-carboxylase, can be related to: 1) Cu-induced hepatotoxicity (cytochrome oxidase subunit I, alanine aminotransferase, glutathione S-transferase); 2) potential immunosuppression due to interference of Cu with the inflammation/immunity network (α-1 antitrypsin, angiotensinogen, complement component C3, recombination-activating protein-1 and warm temperature acclimation-related 65 kDa protein).
Fish & Shellfish Immunology 09/2011; 31(6):1051-8. · 2.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Erosive reflux disease (ERD) and non-erosive reflux disease (NERD) are often regarded as part of the spectrum of the same disease.
To elucidate molecular features that characterise NERD and ERD at the protein level.
A total of 56 consecutive subjects were enrolled: 10 healthy subjects, 24 with NERD and 22 with ERD. Eight specimens were taken from macroscopically normal mucosa at 5 cm of gastro-oesophageal junction. Four were processed for the proteins extraction and four for evaluation using haematoxylin-eosin and immunohistochemistry. We used shotgun proteomics to identify tentative disease molecular features for ERD or NERD. Candidate distinctive proteins were verified using immunohistochemistry.
Shotgun proteomics analysis revealed 33 differentially expressed proteins in NERD vs. ERD samples, involved in cellular proliferation, keratinisation, stress responses and sugar metabolism. Based on a gene ontology meta-analysis, seven of them were further analysed using Western blotting (WB) and four also using immunohistochemistry. We identified novel candidate disease molecular features for GERD and few distinctive proteins to discriminate NERD and ERD. In particular, Transitional Endoplasmic Reticulum ATPase (TER ATPase), GAPDH, Alpha 1 Acid Glycoprotein 1, Annexin A1, Calmodulin and 14-3-3 proteins were confirmed at WB analysis.
Non-erosive reflux disease and ERD are distinct disease entities at the protein level. This study proposes an array of candidate biomarkers possibly useful to discriminate between NERD and ERD.
[Show abstract][Hide abstract] ABSTRACT: A PS-b-PMA block copolymer bearing a terminal carboxylic group has been synthesized using 3-(benzylsulfanyl thiocarbonylsulfanyl) propionic acid as RAFT agent. When dispersed in methanol, this block copolymer displays pseudo-amphiphilic behaviour providing micelles which can be suspended in water by osmosis. The carboxylic terminations are fundamental during the self-assembling process contributing to the formation of nanosized monodispersed particles. Furthermore, the anionic corona confers the to micelles stability in aqueous media. The core of the micelles was loaded with Nile Red to achieve the intracellular delivery of a lipophilic substance; this was demonstrated by Confocal Laser Scanning Microscopy. The particles, when loaded with doxorubicin, are able to overcome the intrinsic resistance of LoVo-MDR cancer cells. Further stabilization is given to the micelles by covalent shell crosslinking using triethylene glycol diacrylate. Because of the increased intracellular stability of the crosslinked micelles, they stain, when loaded with Nile Red, the aqueous cytosol instead of the lipophilic compartments. The unimers constituting the micelles were covalently labeled with fluorescein to follow their fate once incorporated into the cells.
Journal of Materials Chemistry 02/2011; 21(8):2555-2562. · 5.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Regulator of calcineurin 3 (RCAN3) belongs to the human RCAN gene family, which also includes RCAN1 and RCAN2. All three members interact with and inhibit calcineurin. Based on this effect, several studies have demonstrated a role for RCAN1 and RCAN2 on inflammation, using human umbilical vein endothelial cells (HUVECs) as a model. RCAN1 and 2 are strongly induced by vascular endothelial growth factor (VEGF), inhibit cell proliferation and down-regulate many pro-inflammatory and pro-angiogenic genes. The present work is the first study to investigate the role of RCAN3 on inflammation in HUVECs. RCAN3 isoforms have been characterized and quantified in HUVECs; only those with the same frame are expressed and show a peculiar expression pattern. RCAN3 inhibits HUVEC proliferation both basally and under VEGF or phorbol 12-myristate 13-acetate-stimulated conditions, however it does not modulate gene expression of the chosen inflammatory genes. Results indicate an interesting role for RCAN3 in modulating HUVEC proliferation, independently from the inflammatory and angiogenic processes.
International Journal of Molecular Medicine 12/2010; 26(6):913-8. · 1.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The exposure of the human body to microgravity, conditions that occurs during space flights, causes significant changes in the cardiovascular system. Many cell types have been involved in these changes, and the endothelium seems to play a major role. In endothelial cells (EC), it has been shown that modeled low gravity impairs nitric oxide synthesis, cell adhesion, extracellular matrix composition, cytoskeleton organization, cytokines, and growth factors secretion. Nevertheless, detailed analysis of EC physiological changes induced by microgravity exposure is still lacking. Secretome analysis is one of the most promising approaches for the identification of biomarkers directly related to the physiopathological cellular state. In this study, we analyzed in details the modifications of EC secretome by using umbilical vein endothelial (HUVE) cells exposed to modeled low gravity conditions. By adopting a two-dimensional (2-D) proteomic approach, in conjunction with a technique for the compression of the dynamic range of proteins, we observed that modeled low gravity exposure of HUVE cells affected the secretion of proteins involved in the regulation of cytoskeleton assembly. Moreover, by using Luminex® suspension array systems, we found that the low gravity condition decreased in ECs the secretion of some key pro-inflammatory cytokines, including IL-1α and IL-8, and of the pro-angiogenic factor bFGF. On the contrary, microgravity increase the secretion of two chemokines (Rantes and Eotaxin), involved in leukocytes recruitment.
Journal of Cellular Biochemistry 11/2010; 112(1):265-72. · 3.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cyclooxygenase-2 (COX-2) overexpression is strongly associated with colorectal tumourigenesis. It has been demonstrated that the chronic use of non-steroidal anti-inflammatory drugs (COX inhibitors) partially protects patients from colorectal cancer (CRC) development and progression but induces severe cardiovascular side effects. New strategies for selective COX-2 blockade are required.
We developed an improved technique, based on RNA interference (RNAi), to gain a selective COX-2 silencing in CRC cells by a tumour-dependent expression of anti-COX-2 short-hairpin RNA (shCOX-2). Anti-COX-2 shRNA-expressing vectors were delivered in CRC cells (in vitro) and in colon tissues (ex vivo) using engineered Escherichia coli strains, capable of invading tumour cells (InvColi).
A highly tumour-dependent shCOX-2 expression and a significant COX-2 silencing were observed in CRC cells following InvColi strain infection. Cyclooxygenase-2 silencing was associated with a strong reduction in both proliferative and invasive behaviour of tumour cells. We also demonstrated a pivotal role of COX-2 overexpression for the survival of CRC cells after bacterial infection. Moreover, COX-2 silencing was achieved ex vivo by infecting colon tissue samples with InvColi strains, leading to anti-inflammatory and anti-tumour effects.
Our RNAi/InvColi-mediated approach offers a promising tool for a highly selective COX-2 blockade in vitro and in vivo.
British Journal of Cancer 09/2010; 103(7):975-86. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cuprizone is used to obtain demyelination in mice. Cuprizone-treated mice show symptoms similar to several neurodegenerative disorders such as severe status spongiosus. Although it has a simple chemical formula, its neurotoxic mechanism is still unknown. In this work, we examined both physico–chemical properties and biological effects of cuprizone. Our results indicate that cuprizone has very complicated and misunderstood solution chemistry. Moreover, we show here the inability of cuprizone to cross neither the intestinal epithelial barrier nor the neuronal cell membrane, as well its high tolerability by cultured neurons. If added to mice diet, cuprizone does not accumulate in liver or in brain. Therefore, its neurotoxic effect is explainable only in terms of its capability to chelate copper, leading to chronic copper deficiency.
[Show abstract][Hide abstract] ABSTRACT: Cannabinoid (CB) receptors have been located in brain areas involved in the triggering of TLESRs as well as in the nodose ganglion from which vagal afferents emanate. The distribution of CB(1) receptors has been investigated in the human gastrointestinal mucosa, as expression of inflammatory process.
To evaluate the CB(1) expression in oesophageal mucosa.
A total of 87 consecutive subjects were enrolled: 10 controls, 39 NERD and 38 erosive oesophagitis. Eight specimens were taken from macroscopically normal mucosa. Five were processed by haematoxylin-eosin, MIB1/CB(1) evaluation and three for the RNA and proteins extraction.
The mean MIB1-LI value was 31% and 22% in NERD and ERD patients, respectively, compared to 68% in the healthy subjects. Mean CB(1)mRNA/GUSB mRNA value of the controls was 0.66, while in GERD patients, it was 0.28. In NERD and ERD, the mean values of CB(1)/GUSB were 0.38 and 0.17, respectively, with highly significant differences between the NERD vs. ERD groups. Semi-quantitative analysis of CB(1) expression, performed with WB, shows in NERD patients a higher CB(1) receptor expression than ERD patients.
With this study, we showed for the first time the presence of CB(1) receptors in the human oesophageal epithelium.
[Show abstract][Hide abstract] ABSTRACT: Cyclooxygenase-2 (COX-2) enzyme has been involved in the tumorigenesis and in the progression of colorectal cancer (CRC). The use of traditional nonsteroidal anti-inflammatory drugs (NSAIDs) or selective COX-2 inhibitors has been proposed for the prevention and the treatment of this relevant neoplastic disease. In the light of an innovative alternative to these pharmacological approaches, we review here the possible strategies to achieve a strong and selective inhibition of COX-2 enzyme by using the mechanism of RNA Interference (RNAi) targeted against its mRNA. Anti-COX-2 siRNA molecules (siCOX-2) can be generated in CRC cells from short hairpin RNA (shRNA) precursors, delivered in vitro by a retroviral expression system, and induce a significant and stable silencing of overexpressed COX-2 in human colon cancer cells. As a safer alternative to viral approach, nonpathogenic bacteria (E. coli) can be engineered to invade eukaryotic cells and to generate siCOX-2 molecules in cancer cells. Moreover, the involvement of miRNAs in COX-2 posttranscriptional regulation opens up the possibility to exploit an endogenous silencing mechanism to knockdown overexpressed COX-2. Thus, these recent strategies disclose new challenging perspectives for the development of clinically compatible siRNA or miRNA capable of selectively inhibiting COX-2 enzyme.
BioMed Research International 01/2010; 2010:828045. · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Development of renal biomarkers is required to improve on diagnostic accuracy, prognosis and prediction of response to therapy in renal disease. We describe a new method of obtaining from renal specimens a biologic fluid potentially enriched in secreted proteins.
A renal biopsy specimen was centrifuged, and the interstitial fluid (IF) obtained was evaluated by SELDI-ToF, 1D and 2D gel electrophoresis. Twelve spots were extracted from the 2D gel and characterized by MALDI-TOF-MS.
The SELDI diagrams demonstrated abundant peptide peaks. One-dimensional gel electrophoresis demonstrated the presence of many bands indicating a diversity of proteins in the sample. Comparison of serum to IF demonstrated a number of bands that were not shared, suggesting that the IF is not a simple "replica" of plasma fluid. Employing 2D-PAGE, 418 spots were identified in the IF sample; 12 spots were selected and analyzed by mass spectrometry.
We have described a novel technique to obtain a biologic fluid that contains a significant quantity and diversity of proteins from renal tissue. The procedure to obtain the fluid is simple and easily applicable to standard renal biopsy procedures. This fluid has the potential to identify informative proteins that are more concentrated than in any other renal biologic fluid previously analyzed and strictly related to renal pathophysiology. Future work includes the development of a clinical protocol to identify and validate informative biomarkers that have diagnostic and prognostic value.
Journal of nephrology 01/2010; 24(3):329-37. · 2.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Copper dyshomeostasis is responsible for the neurological symptoms observed in the genetically inherited copper-dependent disorders (e.g., Menkes' and Wilson's diseases), but it has been also shown to have an important role in neurodegenerative diseases such as Alzheimer disease, prion diseases, Parkinson's disease and amyotrophic lateral sclerosis. It is widely accepted that increased extracellular copper levels contribute to neuronal pathogenic process by increasing the production of dangerous radical oxygen species, but the existence of other molecular mechanisms explaining copper neurotoxicity has not been investigated yet. By using a cellular model based on hypothalamic GN11 cultured neurons exposed to copper supplementation and by analysing the cell conditioned media, we try here to identify new molecular events explaining the association between extracellular copper accumulation and neuronal damages. We show here that increased extracellular copper levels produce a wide complex of alterations in the neuronal extracellular environment. In particular, copper affects the secretion of molecules involved in the protection of neurons against oxidative stress, such as cyclophilin A (CypA), or of molecules capable of shifting neuronal cells towards a pro-inflammatory state, such as IL-1alpha, IL-12, Rantes, neutrophil gelatinase-associated lipocalin (NGAL) and secreted protein acidic and rich in cysteine (SPARC). Copper pro-inflammatory properties have been confirmed by using primary neurons.