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Inflammatory bowel diseases (IBD) are chronic disabling conditions with a complex and multifactorial etiology, which is still not completely understood. In the last 20 years, anti-TNF-α antagonists have revolutionized the treatment of IBD, but many patients still do not respond or experience adverse events. Therefore, new biological therapies and small molecules, targeting several different pathways of gut inflammation, have been developed of which some have already been introduced in clinical practice while many others are currently investigated. Moreover, therapeutic procedures such as leukocytapheresis, fecal microbiota transplant and stem cell transplantation are currently being investigated for treating IBD. Lastly, complementary and alternative medicine has become a field of interest for gastroenterologist to reduce symptom burden in IBD patients. In this comprehensive and updated review, a novel classification of current and developing drugs is provided.

OBJECTIVES Cytokines play a pivotal role in inflammatory bowel disease (IBD). We investigated the expression of inflammatory and regulatory cytokines in inflamed and uninflamed mucosal samples of ulcerative colitis patients. METHODS Twenty-five ulcerative colitis patients were enrolled. Bioptic samples from inflamed and not inflamed intestinal areas were obtained. Multiplex analysis for inflammatory and regulatory cytokines was performed. Serum C-reactive protein (CRP) was assessed. Endoscopic Mayo score and histological simplified Geboes score were calculated. RESULTS Interleukin (IL)-1Ra, IL-6, IL-8, IL-17, induced Protein (IP)-10, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1a, MIP-1b resulted increased in ulcerative colitis inflamed vs ulcerative colitis not inflamed areas. No differences were registered between conventional and anti-tumor necrosis factor-a regimens. No difference with CRP levels was found. IL-7 resulted reduced in patients with endoscopic Mayo score ≥2. All the not inflamed samples had a Geboes score

Marker di staminalità nei carcinomi squamocellulari del cavo orale e orofaringe in relazione al sito di origine e a infezione da HPV: implicazioni cliniche.In questo studio è stata esaminata l’espressione di potenziali marcatori di staminalità nei carcinomi della testa e collo (HNSCC) per valutarne il loro possibile ruolo clinico. Sono stati arruolati 69 carcinomi squamocellulari del cavo orale (OSCC) e dell’orofaringe (OPSCC) primitivi e non precedentemente sottoposti a trattamento, raccogliendo i dati anagrafici, clinici e sul follow up. Abbiamo valutato l’eventuale infezione da HPV e l’espressione di 5 potenziali marker di staminalità (CD44, CD133, Oct-4, Nanog, and Sox-2). Gli OPSCC positivi per HPV hanno mostrato minor espressione di Nanog, mentre la sua espressione citoplasmatica è stata associata con una prognosi significativamente peggiore negli OPSCC ma non in OSCC. La colorazione di Sox-2 si è rivelata più intensa tra gli OPSCC, e la sua espressione nucleare è associata con una peggiore prognosi. L’espressione di Nanog è associata a OPSCC HPV-negativi e può avere un ruolo come marker diagnostico surrogato. In conclusione il profilo di espressione di alcuni marker di cellule staminali nei HNSCC sembra essere differente a seconda del sito di origine del tumore e dell’infezione da HPV. Inoltre Nanog e Sox-2 potrebbero presentare un significato prognostico.The expression of potential stem cell markers in HNSCCs was investigated to assess their potential clinical role. 69 primary, previously untreated oral (OSCC) and oropharyngeal squamous cell carcinomas (OPSCC) were enrolled; personal, clinical and follow-up data were collected. HPV infection and expression of 5 potential stem cell markers (CD44, CD133, Oct-4, Nanog, and Sox-2) were evaluated. HPV+ OPSCC showed lower expression of Nanog. The cytoplasmic expression of Nanog was associated with significantly worse prognosis in OPSCC, but not in OSCC. Sox-2 staining was more intense among OPSCCs. Sox-2 nuclear staining was associated with worse prognosis. Nanog expression was associated with HPV- OPSCC and may have a role as a surrogate diagnostic marker. In general, the expression profile of some stem cell markers in HNSCC seems to vary according to the site of origin and HPV infection. Nanog and Sox-2 may also have prognostic value.

Recurrent cystitis (RC) is a common disease, especially in females. Anatomical, behavioral and genetic predisposing factors are associated with the ascending retrograde route, which often causes bladder infections. RC seems to be mainly caused by agents derived from the intestinal microbiota, and most frequently by Escherichia coli. Intestinal contiguity contributes to the etiopathogenesis of RC and an alteration in intestinal permeability could have a major role in RC. The aim of this pilot study is to assess gut microbiome dysbiosis and intestinal permeability in female patients with RC. Patients with RC (n = 16) were enrolled and compared with healthy female subjects (n = 15) and patients with chronic gastrointestinal (GI) disorders (n = 238). We calculated the Acute Cystitis Symptom Score/Urinary Tract Infection Symptom Assessment (ACSS/UTISA) and Gastrointestinal Symptom Rating Scale (GSRS) scores and evaluated intestinal permeability and the fecal microbiome in the first two cohorts. Patients with RC showed an increased prevalence of gastrointestinal symptoms compared with healthy controls. Of the patients with RC, 88% showed an increased intestinal permeability with reduced biodiversity of gut microbiota compared to healthy controls, and 68% of the RC patients had a final diagnosis of gastrointestinal disease. Similarly, GI patients reported a higher incidence of urinary symptoms with a diagnosis of RC in 20%. Gut barrier impairment seems to play a major role in the pathogenesis of RC. Further studies are necessary to elucidate the role of microbiota and intestinal permeability in urinary tract infections.

Nutritional deficiencies are common in inflammatory bowel diseases (IBD). In patients, magnesium (Mg) deficiency is associated with disease severity, while in murine models, dietary Mg supplementation contributes to restoring mucosal function. Since Mg availability modulates key bacterial functions, including growth and virulence, we investigated whether the beneficial effects of Mg supplementation during colitis might be mediated by gut microbiota. The effects of dietary Mg modulation were assessed in a murine model of dextran sodium sulfate (DSS)-induced colitis by monitoring magnesemia, weight, and fecal consistency. Gut microbiota were analyzed by 16S-rRNA based profiling on fecal samples. Mg supplementation improved microbiota richness in colitic mice, increased abundance of Bifidobacterium and reduced Enterobacteriaceae. KEEG pathway analysis predicted an increase in biosynthetic metabolism, DNA repair and translation pathways during Mg supplementation and in the presence of colitis, while low Mg conditions favored catabolic processes. Thus, dietary Mg supplementation increases bacteria involved in intestinal health and metabolic homeostasis, and reduces bacteria involved in inflammation and associated with human diseases, such as IBD. These findings suggest that Mg supplementation may be a safe and cost-effective strategy to ameliorate disease symptoms and restore a beneficial intestinal flora in IBD patients.

Background Probiotics are defined as live, nonpathogenic bacteria that confer health benefits beyond their nutritional value. In particular, VSL#3 exhibits demonstrated efficacy in the management of diseases characterized by an increased intestinal permeability. Our study aimed to understand how VSL#3 promotes gut health by secreting bioactive factors and identify which human pathways are modulated by secretome derived from the VSL#3 formula. Methods Two different lots of VSL#3 were used, and Caco-2 cell line was treated with conditioned media (CM) prepared using 1 g of the probiotic formula. We evaluated the effects of the probiotics on cellular proliferation and apoptosis by cytometry and the expression of tight junction proteins by western blotting. A proteomics analysis of both culture media and the whole proteome of Caco-2 cells treated with VSL#3-CM was performed by nano-ultra performance liquid chromatography - tandem mass (nUPLC MS/MS) spectrometry. Results The probiotic formula increased cell proliferation, decreased cellular apoptosis cells, and increased re-epithelialization in the scratch assay. Several peptides specifically synthetized by all the species within the probiotic preparation were recognized in the proteomics analysis. Human proteins synthesized by CaCo-2 cells were also identified. Conclusions To our knowledge, this manuscript describes the first evaluation of the probiotic secretome, and the results showed that the improvement in intestinal barrier functions induced by probiotics seems to be accompanied by the modulation of some human cellular pathways.

Background Probiotics are defined as live, non-pathogenic bacteria that confer health benefits beyond their nutritional value. Particularly VSL#3, a probiotic mix containing 4 strains of Lactobacilli (L. paracasei, L. plantarum, L. acidophilus and L. delbrueckii subsp. bulgaricus), 3 strains of Bifidobacteria (B. longum, B. infantis, B. breve) and Streptococcus thermophilus, has demonstrated efficacy in the management of diseases characterized by increased intestinal permeability. The aim of the present study was to study secreted bioactive factors in order to evaluate the mechanisms of action to enhance intestinal epithelia. Methods Two different lots of VSL#3 (Manufacturer: Nutrilinea Srl, Gallarate (VA) - Italy, lot #802097 and lot #802100) were used. Caco2 cell line were treated with a conditioning media (CM) prepared using 1g of probiotic formula grown in D-MEM cell culture medium (free of serum and antibiotics) at 37°C for 48 hours without shaking and in anaerobic conditions. Caco2 were treated with diluted CM at 1:10 and 1:25 for 24 and 48 hours. Media culture for each condition has been collected and analyzed by a deeper proteomics approach. Differential protein expression was evaluated by shotgun proteomics analysis based on nLC-HDMSE and carried out on Synapt G2-Si mass spectrometer. Results The analysis of supernatants from Caco2, treated with CM, showed the presence of bacteria strain-specific proteins. Human proteins synthesized from CaCo2 were also identified, such as caspase 1, IL8, HSP70, HSP70b, HSP90, HSP105. The productions were time- and dose-dependent. In CM diluted 1:10, probiotic-derived proteins have been shown to be more expressed at 24 hours. Human caspase 1, IL8, HSP 70, HSP 70b, HSP 90, HSP 105 were also found upregulated in Caco2 treated for 24 hours with CM diluted 1:10. Conclusions This is the first time where a probiotic secretome was explored. Analysis of secretome from Caco2 treated with CM helped us to understand the mechanism by probiotics can enhance intestinal barrier: by strengthen the autophagy process, an arm of innate immunity, by overexpression of caspase 1, IL8 and HSP 70, and by HSPs dependent modulation of inflammation.

Background Anti-tumor necrosis factor α (TNFα) represents the best therapeutic option to induce mucosal healing and clinical remission in patients with moderate-severe ulcerative colitis. On the other side gut microbiota plays a crucial role in pathogenesis of ulcerative colitis but few information exists on how microbiota changes following anti-TNFα therapy and on microbiota role in mucosal healing. Aim To elucidate whether gut microbiota and immune system changes appear following anti TNFα therapy during dextran sulfate sodium (DSS) colitis. Methods Eighty C57BL/6 mice were divided into four groups: "No DSS", "No DSS + anti-TNFα", "DSS" and "DSS + anti-TNFα". "DSS" and "DSS + anti-TNFα" were treated for 5 d with 3% DSS. At day 3, mice whithin "No DSS+anti-TNFα" and "DSS+anti-TNFα" group received 5 mg/kg of an anti-TNFα agent. Forty mice were sacrificed at day 5, forty at day 12, after one week of recovery post DSS. The severity of colitis was assessed by a clinical score (Disease Activity Index), colon length and histology. Bacteria such as Bacteroides, Clostridiaceae, Enterococcaceae and Fecalibacterium prausnitzii (F. prausnitzii) were evaluated by quantitative PCR. Type 1 helper T lymphocytes (Th1), type 17 helper T lymphocytes (Th17) and CD4+ regulatory T lymphocytes (Treg) distributions in the mesenteric lymph node (MLN) were studied by flow cytometry. Results Bacteria associated with a healthy state (i.e., such as Bacteroides, Clostridiaceae and F. prausnitzii) decreased during colitis and increased in course of anti-TNFα treatment. Conversely, microorganisms belonging to Enterococcaceae genera, which are linked to inflammatory processes, showed an opposite trend. Furthermore, in colitic mice treated with anti-TNFα microbial changes were associated with an initial increase (day 5 of the colitis) in Treg cells and a consequent decrease (day 12 post DSS) in Th1 and Th17 frequency cells. Healthy mice treated with anti-TNFα showed the same histological, microbial and immune features of untreated colitic mice. "No DSS + anti-TNFα" group showed a lymphomononuclear infiltrate both at 5th and 12th d at hematoxylin and eosin staining, an increase of in Th1 and Th17 frequency at day 12, an increase of Enterococcaceae at day 5, a decrease of Bacteroides and Clostridiaceae at day 12. Conclusion Anti-TNFα treatment in experimental model of colitis improves disease activity but it is associated to an increase in Th17 pathway together with gut microbiota alteration.

Aim To explore the influence of Infliximab (IFX) on cancer progression in a murine model of colonic cancer associated to chronic colitis. Methods AOM/DSS model was induced in C57BL/6 mice. Mice were injected with IFX (5 mg/kg) during each DSS cycle while control mice received saline. Body weight, occult blood test and stool consistency were measured to calculate the disease activity index (DAI). Mice were sacrificed at week 10 and colons were analyzed macroscopically and microscopically for number of cancers and degree of inflammation. MTT assay was performed on CT26 to evaluate the potential IFX role on metabolic activity and proliferation. Cells were incubated with TNF-α or IFX or TNF-α plus IFX, and cell vitality was evaluated after 6, 24 and 48 h. The same setting was used after pre-incubation with TNF-α for 24 h. Results IFX significantly reduced DAI and body weight loss in mice compared with controls, preserving also colon length at sacrifice. Histological score was also reduced in treated mice. At macroscopic analysis, IFX treated mice showed a lower number of tumor lesions compared to controls. This was confirmed at microscopic analysis, although differences were not statistically significant. In vitro, IFX treated CT26 maintained similar proliferation ability at MTT test, both when exposed to IFX alone and when associated to TNF-α. Conclusion IFX did not increase colonic cancer risk in AOM-DSS model of cancer on chronic colitis nor influence directly the proliferation of murine colon cancer epithelial cells.

Determination of urinary lactulose/mannitol is one of the most used tests to evaluate intestinal barrier function. High-performance liquid chromatography (HPLC) separation with electrospray ionization tandem mass spectrometry guarantees high levels of selectivity and reproducibility. In this paper we report an upgrade of the previous published liquid chromatography tandem mass spectrometry method, introducing more reliable internal standards and ultra-performance liquid chromatography with ethylene bridged hybrid amide columns. The ultra-performance liquid chromatography provided an efficient chromatographic separation of the two sugars in 5 min, compared to 15 min using the previous method. The limit of quantification was 10 &micro, g/mL for mannitol and 2.5 &micro, g/mL for lactulose, and the assay was linear up to 1000 &micro, g/mL for mannitol and 1000 &micro, g/mL for lactulose. The within-run precision and accuracy ranged from 0.7 to 2.9% and 97.2 to 101.2%, respectively. The between-run precision and accuracy ranged from 1.9 to 4.7% and 94.8 to 97.5%, respectively. Recovery was higher than 90.2% for both lactulose and mannitol, and the matrix effect for both lactulose and mannitol was lower than 15%. With this new method we have a real improvement in terms of accuracy and reproducibility, ensuring results in shorter time. The changes to the previous protocol make this method excellent for routine purposes.

In recent years, there has been an increasing interest on muscle wasting, considering the reduction of quality of life and the increase of morbidity and mortality associated. Sarcopenia and cachexia represent two conditions of reduction of muscle mass, sharing several elements involved in their pathogenesis, such as systemic inflammation, impaired muscle protein synthesis, increased muscle apoptosis, mitochondrial dysfunction in skeletal muscle tissue and insulin resistance. These features often characterize cancer, inactivity or denervation, but also inflammatory diseases, such as chronic obstructive pulmonary disease, renal failure, cardiac failure, rheumatoid arthritis, inflammatory bowel disease and aging in general. The gastrointestinal tract and gut microbiota are thought to be deeply associated with muscle function and metabolism, although the exact mechanisms that link gut with skeletal muscle are still not well known. This review summarized the potential pathways linking gut with muscle, in particular in conditions as sarcopenia and cachexia. The main emerging pathways implicated in the skeletal muscle-gut axis are: the myostatin/activin signaling pathway, the IGF1/PI3K/AKT/mTOR signaling pathway, which results suppressed, the NF-kB signaling pathway and the FOXO signaling pathway. Further researches in this field are necessary to better explain the linkage between gut microbiota and muscle wasting and the possible emerging therapies associated.

Over the last few years, the gut microbiota has been the focus of countless studies conducted both on mouse models and human population, aimed at analyzing its functions and interactions with the host, including nutrition, metabolic homeostasis, protection from infections and development of systemic and mucosal immunity both in inflammatory bowel disease (IBD) as well as other intestinal and extra-intestinal diseases. In IBD microbiota is impaired in overall composition and biodiversity, stability as well as functions. Microbial signature of IBD can be considered also a decrease in F. prausnitzii, increase of Proteonbacteria as well as the described increase of Candida albicans, Basidiomycota/Ascomycota ratio over Saccharomyces cerevisiae and of the Caudovirales over Microviridae. The indirect (through antibiotics, probiotics) and direct (through fecal microbiota transplantation) modulation of gut microbiota has relevant clinical implication in IBD management. In the near future role and clinical implication of gut microbiota characterization in the therapeutic personalized approach to IBD patients will eventually become clear.

Background There is strong evidence that alcoholism leads to dysbiosis in both humans and animals. However, it is unclear how changes in the intestinal microbiota (IM) relate to ethanol (EtOH)-induced disruption of gut-liver homeostasis. We investigated this issue using selectively bred Sardinian alcohol-preferring (sP) rats, a validated animal model of excessive EtOH consumption. Methods Independent groups of male adult sP rats were exposed to the standard, home-cage 2-bottle "EtOH (10% v/v) versus water" choice regimen with unlimited access for 24 h/d (Group Et) for 3 (T1), 6 (T2), and 12 (T3) consecutive months. Control groups (Group Ct) were composed of matched-age EtOH-naive sP rats. We obtained samples from each rat at the end of each experimental time, and we used blood and colon tissues for intestinal barrier integrity and/or liver pathology assessments and used stool samples for IM analysis with 16S ribosomal RNA gene sequencing. Results Rats in Group Et developed hepatic steatosis and elevated serum transaminases and endotoxin/lipopolysaccharide (LPS) levels but no other liver pathological changes (i.e., necrosis/inflammation) or systemic inflammation. While we did not find any apparent alteration of the intestinal colonic mucosa, we found that rats in Group Et exhibited significant changes in IM composition compared to the rats in Group Ct. These changes were sustained throughout T1, T2, and T3. In particular, Ruminococcus, Coprococcus, and Streptococcus were the differentially abundant microbial genera at T3. The KEGG Ortholog profile revealed that IM functional modules, such as biosynthesis, transport, and export of LPS, were also enriched in Group Et rats at T3. Conclusions We showed that chronic, voluntary EtOH consumption induced liver injury and endotoxemia together with dysbiotic changes in sP rats. This work sets the stage for improving our knowledge of the prevention and treatment of EtOH-related diseases.

INTRODUCTION 13C-urea breath test (UBT) is a non-invasive test for detecting active H. pylori infection. Previous studies showed a correlation of delta over baseline (DOB) values with bacterial load, mucosal inflammation and successful eradication. Gender has been shown to affect DOB in children. Aim of our study was to verify whether gender or ethnicity affects DOB in adults. PATIENTS AND METHODS We retrospectively analyzed data of 2922 patients (1024M/1898F mean age 47±15 years) that underwent UBT in our outpatient unit, from October 2015 to October 2016. Patients were divided based on gender and ethnicity; mean DOB values were then compared. RESULTS 686 pts (23.4%, 258M/428F, mean age 45±17 years) of 2922 pts showed a positive UBT. Prevalence of H. pylori infection was significantly higher in males compared to females (29% vs 22%; p=0,03). Females showed a significant higher mean DOB (34±25 vs 27,6±22; p=0,008). A total of 2922 UBT were performed during the study period (F:1898, 65%; M: 1024 35%). The prevalence of H. pylori infection is 32% in those from Eastern Countries, 28% in those from South America and 40% in both those coming from Africa and Asia. We found significantly lower DOB values in Italians compared to non-Italian (mean DOB 36±27 vs 69±32; p

Major advances have occurred in the knowledge of the pathogenesis of inflammatory bowel disease (IBD) over the last decade, and perhaps the most major, and clinically advantageous of these advances has been the discovery of the microbiome as a key multifaceted component of inflammation. The gut microbiome is the largest known group of cells in the body, and is now recognized as an organ in its own right. Initial studies looking at a possible role of bacterial manipulation of the immune system in IBD, looked at identifying a specific bacterial species, and were not representative of a feasible model of inflammation in IBD overall. More recently there has been a shift towards the concept of dysbiosis, and the acceptance that a number of bacterial factors interact with the immune system in order for inflammation to occur. In the present review we will focus on past perspective of the role of microbiota in IBD, current evidences about dysbiosis in IBD and also the main therapeutic modalities to affect IBD by affecting gut microbiota: probiotics, prebiotics, fecal microbiota transplantation and emerging dietary intervention.

Few data exist on differences in gut microbiota composition among principal gastrointestinal (GI) diseases. We evaluated the differences in gut microbiota composition among uncomplicated diverticular disease (DD), irritable bowel syndrome (IBS) and inflammatory bowel diseases (IBD) patients. DD, IBS, and IBD patients along with healthy controls (CT) were enrolled in our Italian GI outpatient clinic. Stool samples were collected. Microbiota composition was evaluated through a metagenomic gene-targeted approach. GI pathology represented a continuous spectrum of diseases where IBD displayed one extreme, while CT displayed the other. Among Phyla, Biplot PC2/PC3 and dendogram plot showed major differences in samples from IBS and IBD. DD resembled species CT composition, but not for Bacteroides fragilis. In IBS, Dialister spp. and then Faecalibacterium prausnitzii were the most representative species. Ulcerative colitis showed a reduced concentration of Clostridium difficile and an increase of Bacteroides fragilis. In Crohn's disease, Parabacteroides distasonis was the most represented, while Faecalibacterium prausnitzii and Bacteroides fragilis were significantly reduced. Each disorder has its definite overall microbial signature, which produces a clear differentiation from the others. On the other hand, shared alterations constitute the “core dysbiosis” of GI diseases. The assessment of these microbial markers represents a parameter that may complete the diagnostic assessment.

Malnutrition is a major complication of inflammatory bowel disease (IBD). This mini review is focusing on main determinants of malnutrition in IBD, the most important components of malnutrition, including lean mass loss and sarcopenia, as an emerging problem. Each one of these components needs to be well considered in a correct nutritional evaluation of an IBD patient in order to build a correct multidisciplinary approach. The review is then focusing on possible instrumental and clinical armamentarium for the nutritional evaluation.

Purpose Human papillomavirus (HPV) 16 infection is associated with oropharyngeal carcinogenesis and is likely the cause of the reported increase in disease incidence. We evaluated the prevalence of HPV infection and the reliability of different diagnostic tools using primary tumor samples from a cohort of 50 patients. Methods and Materials Formalin-fixed paraffin-embedded (FFPE) tumor samples were collected from all 50 consecutive primary oropharyngeal SCC patients who were enrolled in the study; fresh tumor samples were available in 22 cases. NucliSENS EasyQ HPVv1 was used for RNA, and Digene Hybrid Capture-2(HC2) was used for DNA detection. p16 Expression was evaluated by immunohistochemistry in FPPE specimens. Results Based on the DNA detection assay on FFPE samples, the frequency of high-risk HPV infection was 32%. The agreement rate between HPV RNA and HPV DNA detection in fresh samples was 100%. The agreement rate between p16 immunohistochemistry (IHC) and the detection of HPV DNA in the FFPE samples was fair but not excellent (κ = 0.618). HPV DNA detection was highly significant, as measured by disease-specific survival and determined using a Wilcoxon test ( P =.001). p16 IHC also exhibited a prognostic value but with a lower statistical significance ( P =.0475). The detection of HPV DNA, but not p16 IHC, was also significantly correlated with locoregional control ( P =.0461). Conclusion Diagnostic methods based on the detection of HPV nucleic acids appear to be more reliable and objective because they do not require reading by a trained histopathologist. Furthermore, the detection of HPV DNA exhibits an improved correlation with survival, and therefore appears definitely more reliable than p16 IHC for routine use in clinical practice.

Background Probiotics are defined as live, non-pathogenic bacteria that confer health benefits beyond their nutritional value. Particularly VSL#3, a probiotic mix containing four strains of Lactobacilli (L. paracasei, L. plantarum, L. acidophilus, and L. delbrueckii subsp. bulgaricus*), three strains of Bifidobacteria (B. longum**, B. infantis**, B. breve) and Streptococcus thermophilus, has demonstrated efficacy in the management of diseases characterised by increased intestinal permeability such as irritable bowel syndrome and ulcerative colitis. The aim of the present study was to evaluate the mechanism of action of VSL#3 to reduce intestinal permeability, and its effect in modulating the activity on tight junctions, focusing on the effects of secreted bioactive factors. *Recently reclassified as L. helveticus. **Recently reclassified as B. animalis subsp. lactis. Methods Two different lots of VSL#3 (Manufacturer: Nutrilinea Srl, Gallarate (VA), Italy) were used. Caco2 and HT29 cell lines were treated with a conditioning media (CM) prepared using 1g of probiotic formula grown in D-MEM cell culture medium (free of serum and antibiotics) at 37°C for 48 h without shaking and in anaerobic conditions. The effects of probiotic on proliferations of cells were evaluated by cell growth curve and MTT assay. The effect on apoptosis will be analysed by cytometry using double staining with Annexin V-FITC and Propidium Iodide, as well as it is well established. Furthermore, the effect on expression of tight junctions, in particular claudin-2, occludin, and ZO-1, will be investigated by western blot analysis. Results The multistrain probiotic formula, in particular the more concentrate CM (1:10) increase Caco2 and HT29 cells proliferation and the percentage of metabolic cell activity (up 20%), together with a decrease of 90% of apoptotic cells in the presence of CM. These trophic effects may have an impact on paracellular permeability. The expression of tight junction proteins increase in 24 h for claudin and ZO-1, and after 48 h of treatment in case of occludin. Also the densitometry and the ratio between of relative density of protein and endogenous control (β-actin) confirms these data. The results are the same using two different lots. Conclusions These preliminary data in vitro could be able to explain how VSL#3 works at intestinal mucosa level, in particular by secretion of factors that enhances barrier integrity. The proliferative stimuli and the increase of expression of tight junction proteins are consistent with an effect on mucosa regeneration and re-epithelization. Other experiments, also on other intestinal cell line, are necessary to deeply understand the mechanism of action of this kind of probiotic formula.

Although angiogenesis is viewed as a fundamental component of inflammatory bowel disease (IBD) pathogenesis, we presently lack a thorough knowledge of the cell type(s) involved in its induction and maintenance in the inflamed intestinal mucosa. This study aimed to determine whether platelet (PLT) adhesion to inflamed intestinal endothelial cells of human origin may favour angiogenesis. Unstimulated or thrombin-activated human PLT were overlaid on resting or tumour necrosis factor (TNF)-α-treated human intestinal microvascular endothelial cells (HIMEC), in the presence or absence of blocking antibodies to either vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, integrin α(v) β(3) , tissue factor (TF) or fractalkine (FKN). PLT adhesion to HIMEC was evaluated by fluorescence microscopy, and release of angiogenic factors (VEGF and soluble CD40L) was measured by ELISA. A matrigel tubule formation assay was used to estimate PLT capacity to induce angiogenesis after co-culturing with HIMEC. TNF-α up-regulated ICAM-1, α(v) β(3) and FKN expression on HIMEC. When thrombin-activated PLT were co-cultured with unstimulated HIMEC, PLT adhesion increased significantly, and this response was further enhanced by HIMEC activation with TNF-α. PLT adhesion to HIMEC was VCAM-1 and TF independent but ICAM-1, FKN and integrin α(v) β(3) dependent. VEGF and sCD40L were undetectable in HIMEC cultures either before or after TNF-α stimulation. By contrast, VEGF and sCD40L release significantly increased when resting or activated PLT were co-cultured with TNF-α-pre-treated HIMEC. These effects were much more pronounced when PLT were derived from IBD patients. Importantly, thrombin-activated PLT promoted tubule formation in HIMEC, a functional estimate of their angiogenic potential. In conclusion, PLT adhesion to TNF-α-pre-treated HIMEC is mediated by ICAM-1, FKN and α(v) β(3) , and is associated with VEGF and sCD40L release. These findings suggest that inflamed HIMEC may recruit PLT which, upon release of pro-angiogenic factors, actively contribute to inflammation-induced angiogenesis.

Introduction Infliximab is an effective treatment for inflammatory bowel disease (IBD). Studies differ regarding the influence of body mass index (BMI) on the response to infliximab, with the majority of studies indicating that increased BMI may be associated with a poorer response to Infliximab. However, the pharmacokinetic mechanisms causing this have not yet been reported. Aims Examine the correlation between BMI/immunosuppressant use with clinical response, trough and post-infusion levels of infliximab, tumour necrosis factor-α(TNF-α) and anti-drug antibodies(ATI), and determine if these factors can predict future response. Methods We collected serum from 24 patients receiving Infliximab before and 30 minutes following infusion. Clinical parameters were collected retrospectively and prospectively. ELISA measurements of infliximab, TNF-α and ATI were performed. Results We confirmed that patients with higher infliximab trough levels have a better response rate and that patients with an elevated BMI display a higher rate of loss of response (20%). Patients with a higher BMI had elevated post-infusion levels of infliximab. Additionally, the ratio of IFX/TNF-α trough levels correlated with clinical response to the following infusion. Conclusion This study confirms that an elevated BMI is associated with a poorer response to infliximab. For the first time, we describe that a higher BMI correlates with higher post-infusion levels, however this does not correlate with a higher rate of response to the drug, suggesting that circulating drug levels do not correlate with tissue levels. Furthermore, in our small cohort of patients, we identified a possible predictive marker of future response to treatment which may be used to guide dose escalation and predict non-response to infliximab.

Inflammatory bowel disease (IBD) is an immune-mediated inflammatory condition causing inflammation of gastrointestinal and systemic cells, with an increasing prevalence worldwide. Many factors are known to trigger and maintain inflammation in IBD including the innate and adaptive immune systems, genetics, the gastrointestinal microbiome and several environmental factors. Our knowledge of the involvement of the immune system in the pathophysiology of IBD has advanced rapidly over the last two decades, leading to the development of several immune-targeted treatments with a biological source, known as biologic agents. The initial focus of these agents was directed against the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) leading to dramatic changes in the disease course for a proportion of patients with IBD. However, more recently, it has been shown that a significant proportion of patients do not respond to anti-TNF-α directed therapies, leading a shift to other inflammatory pathways and targets, including those of both the innate and adaptive immune systems, and targets linking both systems including anti-leukocyte trafficking agents-integrins and adhesion molecules. This review briefly describes the molecular basis of immune based gastrointestinal inflammation in IBD, and then describes how several current and future biologic agents work to manipulate these pathways, and their clinical success to date.