Concise Review: Cellular Therapies: The Potential to Regenerate and Restore Tolerance in Immune-Mediated Intestinal Diseases.

Chronic inflammatory enteropathies, including celiac disease, Crohn's disease, and ulcerative colitis, are lifelong disabling conditions whose cure is still an unmet need, despite the great strides made in understanding their complex pathogenesis. The advent of cellular therapies, mainly based on the use of stem cells, represents a great step forward thanks to their multitarget strategy. Both hematopoietic stem cells (HSC) and mesenchymal stem/stromal cells (MSC) have been employed in the treatment of refractory cases with promising results. The lack of immunogenicity makes MSC more suitable for therapeutic purposes as their infusion may be performed across histocompatibility locus antigen barriers without risk of rejection. The best outcome has been obtained when treating fistulizing Crohn's disease with local injections of MSC. In addition, both HSC and MSC proved successful in promoting regeneration of intestinal mucosa, and favoring the expansion of a T-cell regulatory subset. By virtue of the ability to favor mucosal homeostasis, this last cell population has been exploited in clinical trials, with inconsistent results. Finally, the recent identification of the epithelial stem cell marker has opened up the possibility of tissue engineering, with an array of potential applications for intestinal diseases. However, the underlying mechanisms of action of these interconnected therapeutic strategies are still poorly understood. It is conceivable that over the next few years their role will become clearer as the biological interactions with injured tissues and the hierarchy by which they deliver their action are unraveled through a continuous moving from bench to bedside and vice versa. Stem Cells 2016;34:1474-1486.

Validation of shortened 2-day sterility testing of mesenchymal stem cell-based therapeutic preparation on an automated culture system.

Cell therapy products represent a new trend of treatment in the field of immunotherapy and regenerative medicine. Their biological nature and multistep preparation procedure require the application of complex release criteria and quality control. Microbial contamination of cell therapy products is a potential source of morbidity in recipients. The automated blood culture systems are widely used for the detection of microorganisms in cell therapy products. However the standard 2-week cultivation period is too long for some cell-based treatments and alternative methods have to be devised. We tried to verify whether a shortened cultivation of the supernatant from the mesenchymal stem cell (MSC) culture obtained 2 days before the cell harvest could sufficiently detect microbial growth and allow the release of MSC for clinical application. We compared the standard Ph. Eur. cultivation method and the automated blood culture system BACTEC (Becton Dickinson). The time to detection (TTD) and the detection limit were analyzed for three bacterial and two fungal strains. The Staphylococcus aureus and Pseudomonas aeruginosa were recognized within 24 h with both methods (detection limit ~10 CFU). The time required for the detection of Bacillus subtilis was shorter with the automated method (TTD 10.3 vs. 60 h for 10-100 CFU). The BACTEC system reached significantly shorter times to the detection of Candida albicans and Aspergillus brasiliensis growth compared to the classical method (15.5 vs. 48 and 31.5 vs. 48 h, respectively; 10-100 CFU). The positivity was demonstrated within 48 h in all bottles, regardless of the size of the inoculum. This study validated the automated cultivation system as a method able to detect all tested microorganisms within a 48-h period with a detection limit of ~10 CFU. Only in case of B. subtilis, the lowest inoculum (~10 CFU) was not recognized. The 2-day cultivation technique is then capable of confirming the microbiological safety of MSC and allows their timely release for clinical application.

The Circulating Level of Soluble Receptor for Advanced Glycation End Products Displays Different Patterns in Ulcerative Colitis and Crohn's Disease: A Cross-Sectional Study.

BACKGROUND: RAGE is a transmembrane receptor expressed on immune and endothelial cells, whose binding with its ligands, the S100 calgranulins, leads to chronic inflammation. Conversely, its soluble form (sRAGE) plays a protective role by acting as a decoy. We carried out a cross-sectional analysis of the sRAGE and S100A12 serum levels in patients with Crohn's disease (CD) and ulcerative colitis (UC) and searched for a correlation with clinical and biological markers of activity. METHODS: We enrolled 60 CD, 67 UC patients, and 66 controls (all adults). Disease activity was scored through the clinical, endoscopic, and histologic indexes of severity, whilst disease location and behaviour were assessed according to the Montreal classification. In all cases, the levels of serum sRAGE, S100A12, C-reactive protein, and faecal calprotectin were measured. RESULTS: sRAGE levels were significantly lower in UC, both active and inactive, than in controls and CD (817.35, range 437.3-1449; 1211, range 843.7-1618; 1207.5, range 743.15-1875.75; P < 0.05 for both), and inversely correlated with clinical and endoscopic indexes of activity in both IBD groups (P < 0.05 for all) and with the histologic score in the CD group. Moreover, those CD patients with a penetrating behaviour showed a significant reduction in both sRAGE (P = 0.006) and S100A12 (P = 0.034) as compared to those with an inflammatory/stricturing pattern. Although S100A12 levels were not found up-regulated, a negative correlation appeared evident with the clinical (r = -0.38) and endoscopic (r = -0.32) indexes of activity in UC and CD, respectively. CONCLUSION: These data suggest a different role for RAGE in CD and UC, and a potential use of sRAGE as a new biomarker.

Impact of platelet phenotype on myocardial infarction.

In acute myocardial infarction patients the injured vascular wall triggers thrombus formation in the damage site. Fibrin fibers and blood cellular elements are the major components of thrombus formed in acute occlusion of coronary arteries. It has been established that the initial thrombus is primarily composed of activated platelets rapidly stabilized by fibrin fibers. This review highlights the role of platelet membrane phenotype in pathophysiology of myocardial infarction. Here, we regard platelet phenotype as quantitative and qualitative parameters of the plasma membrane outer surface, which are crucial for platelet participation in blood coagulation, development of local inflammation and tissue repair.

Are stem cells a potential therapeutic tool in coeliac disease?

Despite the growing understanding of its pathogenesis, the treatment of coeliac disease is still based on a lifelong gluten-free diet that, although efficacious, is troublesome for affected patients, and a definitive cure is still an unmet need. In this regard, the development of new chemical- and biological-derived agents has often resulted in unsatisfactory effects when tested in vivo, probably because of their ability to target only a single pathway, whilst the immunological cascade responsible for tissue injury is complex and redundant. The advent of cellular therapies, mainly based on the use of stem cells, is an emerging area of interest since it has the advantage of a multi-target strategy. Both haematopoietic and mesenchymal stem cells have been employed in the treatment of refractory patients suffering from autoimmune diseases, with promising results. However, the lack of immunogenicity makes mesenchymal stem cells more suitable than their haematopoietic counterpart, since their transplantation may be performed in the absence of a myeloablative conditioning regimen. In addition, mesenchymal stem cells have been shown to harbour strong modulatory effects on almost all cells involved in immune response, together with a potent regenerative action. It is therefore conceivable that over the next few years their therapeutic use will increase as their biological interactions with injured tissues become clearer.

Tolerogenic effect of mesenchymal stromal cells on gliadin-specific T lymphocytes in celiac disease.

BACKGROUND AIMS: Celiac disease is caused by a dysregulated immune response toward dietary gluten, whose only treatment is a lifelong gluten-free diet. We investigated the effects of mesenchymal stromal cells (MSCs) on gliadin-specific T cells, which are known to induce intestinal lesions, in view of a possible use as new therapy. METHODS: Bone marrow-derived MSCs and gliadin-specific T-cell lines were obtained from allogeneic donors and mucosal specimens of celiac patients, respectively. The immunosuppressant effect of MSCs was evaluated in terms of proliferative response and interferon (IFN)-γ production upon gliadin stimulation of long-term T-cell lines; the immunomodulant effect was assessed in terms of apoptotic rate, immunophenotype and cytokine profile of short-term T-cell lines generated in the presence of MSCs. Different MSC:T-cell ratios were applied, and statistics were performed as appropriate. RESULTS: MSCs inhibited both proliferative response and IFN-γ production of long-term T-cell lines in a dose-dependent manner while limiting the expansion of short-term T-cell lines by increasing the apoptotic rate. Moreover, a reduction of the CD4(+) population and expansion of the regulatory FoxP3+ subset were found in T-cell lines cultured with MSCs, in which a significant decrease of interleukin (IL)-21, IFN-γ and IL-10 paralleled by an upregulation of transforming growth factor-ß1, IL-6 and IL-8 were observed. Finally, an increase of the indoleamine 2,3-dioxygenase activity was found, possibly playing a key role in mediating these effects. CONCLUSIONS: MSCs exert potent immunomodulant effects on gliadin-specific T cells, which may be exploited for future therapeutic application in celiac disease.

Role of the advanced glycation end products receptor in Crohn's disease inflammation.

AIM: To investigate the level of mucosal expression and the involvement of the receptor for the advanced glycation end products (RAGE) in delayed apoptosis and tumor necrosis factor (TNF)-α production in Crohn's disease (CD). METHODS: Surgical and endoscopic specimens from both inflamed and non-inflamed areas of the ileum and/or colon were collected from 20 and 14 adult CD patients, respectively, and used for the assessment of RAGE expression by means of immunohistochemistry and western blotting analysis. Normal tissues from 21 control subjects were used for comparison. The same polyclonal anti-human RAGE antibody (R and D System) was used in all experimental conditions. RAGE staining was quantized by a score including both the amount of positive cells and intensity of immunoreactivity; cellular pattern was also described. The effects of RAGE blocking on apoptotic rate and TNF-α production were investigated on immune cells freshly isolated from CD mucosa and incubated both with and without the muramyl dipeptide used as antigenic stimulus. Statistical analysis was performed via the test for trend, with regression models to account for intra-patient correlations. A 2-sided P < 0.05 was considered significant. RESULTS: In inflamed areas, RAGE expression in both the epithelial and lamina propria compartments was higher than control tissues (P = 0.001 and 0.021, respectively), and a cluster of positive cells were usually found in proximity of ulcerative lesions. Similar results were obtained in the lamina propria compartment of non-inflamed areas (P = 0.025). The pattern of staining was membranous and granular cytosolic at the epithelial level, while in the lamina propria it was diffuse cytosolic. When evaluating the amount of protein expression by immunoblotting, a significant increase of both surface area and band intensity (P < 0.0001 for both) was observed in CD inflamed areas compared to control tissue, while in non-inflamed areas a significant increase was found only for band intensity (P < 0.005). Moreover, a significantly lower expression in non-inflamed areas in comparison with inflamed areas was found for both surface area and band intensity (P < 0.0006 for both). Finally, RAGE blocking largely affects both the apoptotic rate of mucosal cells (towards an increase in both non-inflamed and inflamed areas of P < 0.001 and < 0.0001, respectively) and TNF-α secretion (towards a decrease in both non-inflamed and inflamed areas of P < 0.05 and < 0.01, respectively), mainly in the presence of antigenic stimulation. CONCLUSION: RAGE is up-regulated in CD, especially in inflamed areas, and it appears to play a role in the mechanisms involved in chronic inflammation.