Diletta Arcidiacono

Systemic administration of a novel human umbilical cord mesenchymal stem cells population accelerates the resolution of acute liver injury

BackgroundHepatocytes and stem cells transplantation may be an alternative to liver transplantation in acute or chronic liver disease. We aimed to evaluate the therapeutic potential of mesenchymal stem cells from human umbilical cord (UCMSCs), a readily available source of mesenchymal stem cells, in the CCl4-induced acute liver injury model.MethodsMesenchymal stem cells profile was analyzed by flow cytometry. In order to evaluate the capability of our UCMSCs to differentiate in hepatocytes, cells were seeded on three different supports, untreated plastic support, MatrigelTM and human liver acellular matrix. Cells were analyzed by immunocitochemistry for alpha-fetoprotein and albumin expression, qPCR for hepatocyte markers gene expression, Periodic Acid-Schiff staining for glycogen storage, ELISA for albumin detection and colorimetric assay for urea secretion.To assess the effects of undifferentiated UCMSCs in hepatic regeneration after an acute liver injury, we transplanted them via tail vein in mice injected intraperitoneally with a single dose of CCl4. Livers were analyzed by histological evaluation for damage quantification, immunostaining for Kupffer and stellate cells/liver myofibroblasts activation and for UCMSCs homing. Pro- and anti-inflammatory cytokines gene expression was evaluated by qPCR analysis and antioxidant enzyme activity was measured by catalase quantification.Data were analyzed by Mann–Whitney U-test, Kruskal-Wallis test and Cuzick’s test followed by Bonferroni correction for multiple comparisons.ResultsWe have standardized the isolation procedure to obtain a cell population with hepatogenic properties prior to in vivo transplantation. When subjected to hepatogenic differentiation on untreated plastic support, UCMSCs differentiated in hepatocyte-like cells as demonstrated by their morphology, progressive up-regulation of mature hepatocyte markers, glycogen storage, albumin and urea secretion. However, cells seeded on 3D-supports showed a minor or negligible differentiation capacity.UCMSCs-transplanted mice showed a more rapid damage resolution, as shown by histological analysis, with a lower inflammation level and an increased catalase activity compared to CCl4-treated mice.ConclusionsOur findings show that UCMSCs can be reliably isolated, have hepatogenic properties and following systemic administration are able to accelerate the resolution of an acute liver injury without any differentiation and manipulation. These features make UCMSCs strong candidates for future application in regenerative medicine for human acute liver disease.

The Vibrio cholerae cytolysin promotes chloride secretion from intact human intestinal mucosa.

Background The pathogenicity of the Vibrio cholerae strains belonging to serogroup O1 and O139 is due to the production of virulence factors such as cholera toxin (CT) and the toxin-coregulated pilus (TCP). The remaining serogroups, which mostly lack CT and TCP, are more frequently isolated from aquatic environmental sources than from clinical samples; nevertheless, these strains have been reported to cause human disease, such as sporadic outbreaks of watery diarrhoea and inflammatory enterocolitis. This evidence suggested the possibility that other virulence factor(s) than cholera toxin might be crucial in the pathogenesis of Vibrio cholerae-induced diarrhoea, but their nature remains unknown. VCC, the hemolysin produced by virtually all Vibrio cholerae strains, has been proposed as a possible candidate, though a clear-cut demonstration attesting VCC as crucial in the pathogenesis of Vibrio cholerae-induced diarrhoea is still lacking. Methodology/Principal Findings Electrophysiological parameters and paracellular permeability of stripped human healthy colon tissues, obtained at subtotal colectomy, mounted in Ussing chamber were studied in the presence or absence of VCC purified from culture supernatants of V. cholerae O1 El Tor strain. Short circuit current (ISC) and transepithelial resistance (RT) were measured by a computerized voltage clamp system. The exposure of sigmoid colon specimens to 1 nM VCC resulted in an increase of ISC by 20.7%, with respect to the basal values, while RT was reduced by 12.3%. Moreover, increase in ISC was abolished by bilateral Cl− reduction. Conclusion/Significance Our results demonstrate that VCC, by forming anion channels on the apical membrane of enterocytes, triggers an outward transcellular flux of chloride. Such an ion movement, associated with the outward movement of Na+ and water, might be responsible for the diarrhoea caused by the non-toxigenic strains of Vibrio cholerae.

Umbilical cord mesenchymal stem cells modulate dextran sulfate sodium induced acute colitis in immunodeficient mice.

IntroductionInflammatory bowel diseases (IBD) are complex multi-factorial diseases with increasing incidence worldwide but their treatment is far from satisfactory. Unconventional strategies have consequently been investigated, proposing the use of cells as an effective alternative approach to IBD. In the present study we examined the protective potential of exogenously administered human umbilical cord derived mesenchymal stem cells (UCMSCs) against Dextran Sulfate Sodium (DSS) induced acute colitis in immunodeficient NOD.CB17-Prkdcscid/J mice with particular attention to endoplasmic reticulum (ER) stress.MethodsUCMSCs were injected in NOD.CB17-Prkdcscid/J via the tail vein at day 1 and 4 after DSS administration. To verify attenuation of DSS induced damage by UCMSCs, Disease Activity Index (DAI) and body weight changes was monitored daily. Moreover, colon length, histological changes, myeloperoxidase and catalase activities, metalloproteinase (MMP) 2 and 9 expression and endoplasmic reticulum (ER) stress related proteins were evaluated on day 7.ResultsUCMSCs administration to immunodeficient NOD.CB17-Prkdcscid/J mice after DSS damage significantly reduced DAI (1.45 ± 0.16 vs 2.08 ± 0.18, p < 0.05), attenuating the presence of bloody stools, weight loss, colon shortening (8.95 ± 0.33 cm vs 6.8 ± 0.20 cm, p < 0.01) and histological score (1.97 ± 0.13 vs 3.27 ± 0.13, p < 0.001). Decrease in neutrophil infiltration was evident from lower MPO levels (78.2 ± 9.7 vs 168.9 ± 18.2 U/g, p < 0.01). DSS treatment enhanced MMP2 and MMP9 activities (>3-fold), which were significantly reduced in mice receiving UCMSCs. Moreover, positive modulation in ER stress related proteins was observed after UCMSCs administration.ConclusionsOur results demonstrated that UCMSCs are able to prevent DSS-induced colitis in immunodeficient mice. Using these mice we demonstrated that our UCMSCs have a direct preventive effect other than the T-cell immunomodulatory properties which are already known. Moreover we demonstrated a key function of MMPs and ER stress in the establishment of colitis suggesting them to be potential therapeutic targets in IBD treatment.

Detection of fluorescent organic nanoparticles by confocal laser endomicroscopy in a rat model of Barrett’s esophageal adenocarcinoma

For many years, novel strategies for cancer detection and treatment using nanoparticles (NPs) have been developed. Esophageal adenocarcinoma is the sixth leading cause of cancer-related deaths in Western countries, and despite recent advances in early detection and treatment, its prognosis is still very poor. This study investigated the use of fluorescent organic NPs as potential diagnostic tool in an experimental in vivo model of Barrett’s esophageal adenocarcinoma. NPs were made of modified polysaccharides loaded with [4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran] (DCM), a well-known fluorescent dye. The NP periphery might or might not be decorated with ASYNYDA peptide that has an affinity for esophageal cancer cells. Non-operated and operated rats in which gastroesophageal reflux was surgically induced received both types of NPs (NP-DCM and NP-DCM-ASYNYDA) by intravenous route. Localization of mucosal NPs was assessed in vivo by confocal laser endomicroscopy, a technique which enables a “real time” and in situ visualization of the tissue at a cellular level. After injection of NP-DCM and NP-DCM-ASYNYDA, fluorescence was observed in rats affected by esophageal cancer, whereas no signal was observed in control non-operated rats, or in rats with simple esophagitis or Barrett’s esophagus mucosa. Fluorescence was observable in vivo 30 minutes after the administration of NPs. Interestingly, NP-DCM-ASYNYDA induced strong fluorescence intensity 24 hours after administration. These observations suggested that NPs could reach the tumor cells, likely by enhanced permeability and retention effect, and the peptide ASYNYDA gave them high specificity for esophageal cancer cells. Thus, the combination of NP platform and confocal laser endomicroscopy could play an important role for highlighting esophageal cancer conditions. This result supports the potential of this strategy as a targeted carrier for photoactive and bioactive molecules in esophageal cancer diagnosis and treatment.

The Vibrio cholerae cytolysin promotes activation of mast cell (T helper 2) cytokine production.

Many strains of Vibrio cholerae produce a cytolysin (VCC) that forms oligomeric transmembrane pores responsible for vacuolization of several cell types in culture. Here we suggest that VCC could contribute to the T helper 2 (Th2) response seen in the natural infection; acting through TLR2, VCC enhances mast cells secretion of IL‐4, IL‐6 and TNF‐α by 330‐, 290‐ and 550‐fold respectively. Moreover, VCC‐induced cytokine production is dependent on increased cytosolic Ca2+ and on the presence of the Src family kinases Lyn and Fyn, known to be required for FcεRI‐dependent activation of mast cells. These findings strongly suggest that VCC has a pro‐inflammatory activity promoting a Th2‐type immune profile.

Early miR-223 Upregulation in Gastroesophageal Carcinogenesis

Objectives To test miR-223 upregulation during gastric (intestinal-type) and Barrett esophageal carcinogenesis. Methods miR-223 expression was assessed by quantitative reverse transcription polymerase chain reaction in a series of 280 gastroesophageal biopsy samples representative of the whole spectrum of phenotypic changes involved in both carcinogenetic cascades. The results were further validated by in situ hybridization on multiple tissue specimens obtained from six surgically treated gastroesophageal adenocarcinomas. miR-223 expression was also assessed in plasma samples from 30 patients with early stage (ie, stages I and II) gastroesophageal adenocarcinoma and relative controls. Results In both gastric and esophageal models, miR-223 expression significantly increased along with the severity of the considered lesions (analysis of variance, P  <   .001). Among atrophic gastritis and long-segment Barrett esophagus samples, miR-223 overexpression was significantly associated with the score of intestinal metaplasia. miR-223 plasma levels were significantly upregulated in patients with cancer compared with controls ( t test, both P  <   .001). Conclusions miR-223 early upregulation observed in tissue samples and its diagnostic value in discriminating patients with early adenocarcinoma by plasma testing provide a solid rationale for further exploring the diagnostic reliability of this microRNA as a novel biomarker in gastroesophageal adenocarcinoma secondary prevention strategies.

Insulin promotes HER2 signaling activation during Barrett’s Esophagus carcinogenesis

BACKGROUND Insulin-resistance and hyperinsulinemia could have a role in the growing incidence of esophageal adenocarcinoma (EAC) and its pre-cancerous lesion Barretts Esophagus (BE). HER2 activation has also a pivotal role in EAC carcinogenesis but no data correlate these two phenomena in this disease context. AIMS To investigate the role of hyperinsulinemia in BE-dysplasia-adenocarcinoma sequence and the possible relationship between insulin-mediated and HER2 signaling in EAC development. METHODS Serum insulin, C-peptide, IGF1, glucagon, IL-6, TNF-alpha, leptin, adiponectin and Insulin-Resistance-index were analyzed in 19 patients with gastro-esophageal reflux disease, 51 with BE, 24 with dysplastic-BE and 14 with EAC. Insulin/IGF1/HER2 pathways were analyzed in esophageal biopsies using Luminex® Technology. Insulin effect was also evaluated in EAC-derived OE19 cells. Data were analyzed by Fishers exact test, Kruskal-Wallis test, Mann-Whitney U-test, Cuzicks test and Spearman correlation coefficient calculation. RESULTS Insulin-Resistance-index, insulin and C-peptide levels increased along with disease progression (p=0.019, p=0.002, p<0.0001, respectively) and correlated with HER2 expression and with downstream mediators phospho-Akt and phospho-mTOR in esophageal tissue. In vitro, insulin was also able to induce cell proliferation through HER2 activation. CONCLUSIONS Our data pinpoint a possible role of hyperinsulinemia in the Barretts Esophagus metaplasia-dysplasia-adenocarcinoma sequence through HER2 activation in esophageal epithelial cells.

Sex-dependent differences in inflammatory responses during liver regeneration in a murine model of acute liver injury

A sexual dimorphism in liver inflammation and repair was previously demonstrated. Its cellular dissection in the course of acute liver injury (ALI) was explored. BALB/c mice were treated with carbon tetrachloride (CCl4) by intraperitoneal injection and killed after 3, 5, and 8 days. Histological and hepatic cell population analyses were performed. The correlation between androgen receptor (AR) expression and liver recruited inflammatory cells was investigated by treatment with the AR antagonist flutamide. Additionally, patients with a diagnosis of drug induced liver injury (DILI) were included in the study, with a particular focus on gender dimorphism in circulating monocytes. A delayed resolution of necrotic damage and a higher expression of proinflammatory cytokines were apparent in male mice along with a slower recruitment of inflammatory monocytes. F4/80+CD11b+ macrophages and CD11bhighGr-1high monocytes expressed AR and were recruited later in male compared with female livers after CCl4 treatment. Moreover, CD11bhighAR+Gr-1high recruitment was negatively modulated by flutamide in males. Analysis of DILI patients showed overall a significant reduction in circulating mature monocytes compared with healthy subjects. More interestingly, male patients had higher numbers of immature monocytes compared with female patients.A stronger cytotoxic tissue response was correlated with an impaired recruitment of CD11bhighAR+Gr-1high cells and F4/80+CD11b+ macrophages in the early inflammatory phase under AR signaling. During DILI, a dimorphic immune response was apparent, characterized by a massive recruitment of monocytes to the liver both in males and females, but only in males was this recruitment sustained by a turnover of immature monocytes.

Hyperinsulinemia Promotes Esophageal Cancer Development in a Surgically-Induced Duodeno-Esophageal Reflux Murine Model

Hyperinsulinemia could have a role in the growing incidence of esophageal adenocarcinoma (EAC) and its pre-cancerous lesion, Barrett’s Esophagus, a possible consequence of Gastro-Esophageal Reflux Disease. Obesity is known to mediate esophageal carcinogenesis through different mechanisms including insulin-resistance leading to hyperinsulinemia, which may mediate cancer progression via the insulin/insulin-like growth factor axis. We used the hyperinsulinemic non-obese FVB/N (Friend leukemia virus B strain) MKR (muscle (M)-IGF1R-lysine (K)-arginine (R) mouse model to evaluate the exclusive role of hyperinsulinemia in the pathogenesis of EAC related to duodeno-esophageal reflux. FVB/N wild-type (WT) and MKR mice underwent jejunum-esophageal anastomosis side—to end with the exclusion of the stomach. Thirty weeks after surgery, the esophagus was processed for histological, immunological and insulin/Insulin-like growth factor 1 (IGF1) signal transduction analyses. Most of the WT mice (63.1%) developed dysplasia, whereas most of the MKR mice (74.3%) developed squamous cell and adenosquamous carcinomas, both expressing Human Epidermal growth factor receptor 2 (HER2). Hyperinsulinemia significantly increased esophageal cancer incidence in the presence of duodenal-reflux. Insulin receptor (IR) and IGF1 receptor (IGF1R) were overexpressed in the hyperinsulinemic condition. IGF1R, through ERK1/2 mitogenic pattern activation, seems to be involved in cancer onset. Hyperinsulinemia-induced IGF1R and HER2 up-regulation could also increase the possibility of forming of IGF1R/HER2 heterodimers to support cell growth/proliferation/progression in esophageal carcinogenesis.

Mo2008 Ameliorative Potentials of Human Umbilical Cord Derived Mesenchymal Stem Cells in Dextran Sulphate Sodium Induced Acute Colitis in NOD.Cb17-Prkdcscid/J Mice

Introduction: Recent evidences suggest therapeutic value of mesenchymal stem cells (MSCs) as anti-inflammatory and immunomodulatory agent against several autoimmune diseases. Although promising, stem cell based therapies for IBD still requires experimental validation. In the present study we sought to determine whether exogenous administration of human umbilical cord derived cells with phenotype consistent with MSCs (UC-MSCs) can reduce acute colonic damage induced by Dextran Sulphate Sodium (DSS) in immunodeficient NOD.CB17-Prkdcscid/Jmice.Methods: UC-MSCs isolatedwere evaluated by assessing CD73, CD90, CD166, CD105, CD29, CD44, HLA-DR and c-kit expression, by adipocyte, osteocyte differentiation and proliferation potential by cell cycle analysis and wound assay. UC-MSCs were injected in NOD.CB17-Prkdcscid/J mice via tail vein at day 1 and 4. Disease Activity Index, daily body weight changes and colon length, and histological changes evaluated on day 7. Myeloperoxidase, catalase activities, chromosome aberration, sperm head anomaly and metalloproteinase (MMP) 2, 9 expressions were assessed in mice to verify attenuation of DSS induced damage by UC-MSCs. Alteration of ER stress related proteins like BIP, PERK, PDI, CHOP were studied by western blotting. Results: UC-MSC differentiated in osteocytes/ adipocytes, highly expressed CD90, CD73, CD166, CD105 andCD29 typical ofmesenchymal stem cells. UC-MSCs were successfully engrafted in the colon as evident from immunohistochemical staining against anti human nuclei antibody. UC-MSCs administration significantly attenuated bloody stool, weight loss, colon shortening and accelerated healing of damaged mucosa. Decrease in cytotoxicity and genotoxicity was evident from lower MPO level (78.2±9.7 vs 168.9±18.2 U/g, p 3 fold) which were reduced in UC-MSC group. Positive modulation in BIP, PERK, PDI proteins was noticed after MSC administration. Conclusions: NOD.CB17-Prkdcscid/J mice were susceptible to acute DSS colitis, suggesting that T, B and NK cells are not required for the disease. The engrafted UC-MSCs successfully accelerated healing of DSS-induced damaged tissue by promoting tissue repair process by reducing the protease burden through reduced MMP expression in inflammed colon and positively modulating histological score supporting the use of MSCs as a novel therapeutic strategy for colonic injuries. Future works in this direction would hopefully resolve the minutes of this inspiring cell therapy in alleviation of human sufferings involving IBDs.