Pancreatic cancer growth promoted by bone marrow mesenchymal stromal cell-derived IL-6 is reversed predominantly by IL-6 blockade.

Pancreatic cancer is a highly lethal cancer characterized by local invasiveness, early metastasis, recurrence and high resistance to current therapies. Extensive stroma or desmoplasia is a key histological feature of the disease, and interactions between cancer and stromal cells are critical for pancreatic cancer development and progression. Mesenchymal stromal cells [MSCs] exhibit preferential tropism to primary and metastatic tumor sites and may either suppress or support tumor growth. Although MSCs represent a potential source of pancreatic cancer stroma, their contribution to pancreatic tumor growth remains poorly known. Here, we show that bone marrow MSCs significantly contribute to pancreatic cancer growth in vitro and in vivo. Furthermore, MSCs create a pro-carcinogenic microenvironment through the release of key factors mediating growth and angiogenesis, including interleukin (IL)-6, IL-8, vascular endothelial growth factor and activation of STAT3 signaling in tumor cells. IL-6 released by MSCs was largely responsible for the pro-tumorigenic effects of MSCs. Knockdown of IL-6 expression in MSCs by small interfering RNA (siRNA) abolished the MSC growth-promoting effect in vitro, reducing tumor cell proliferation and clonogenic potential. In addition, in a heterotopic nude mouse model of human pancreatic tumor xenografts, blockade of IL-6 with the anti-IL-6 receptor antibody, tocilizumab, or of its downstream effector STAT3 with the small molecule STAT3 inhibitor S3I-201, abrogated MSC-mediated tumor promotion and delayed tumor formation significantly. Our data demonstrate that MSCs promote pancreatic cancer growth, with IL-6 produced by MSCs playing a pivotal role.

Impact of bladder-derived acellular matrix, growth factors, and extracellular matrix constituents on the survival and multipotency of marrow-derived mesenchymal stem cells.

We investigate the effect of bladder-derived acellular matrix (ACM) on bone marrow mesenchymal stem cells (BM-MSC) growth, survival, and differentiation, and evaluate the effect of collagen I and IV on BM-MSC differentiation potential to SMC. BM-MSCs isolated from CD1(_) mice were characterized by surface markers and differentiation into different lineages. BM-MSC SMC potential was further evaluated in stem cell medium alone or supplemented with TGF-ß1 and recombinant human platelet-derived growth factor (PDGF-BB) on plastic, collagen I and IV using western blot. Furthermore, BM-MSCs were seeded on porcine derived ACM-fortified with hyaluronic acid and cultured in Mesencult+-growth factors, bone, or fat induction media for 3 weeks. Seeded constructs were evaluated by H&E, Ki67 assay, Oil red O, and Alizarin red stain. SMC differentiation was semiquantified via immunohistochemistry. BM-MSCs differentiated into fat and bone when induced. In Mesencult, BM-MSCs differentiated into SMC, expressing α-SMA, calponin, and MHC. BM-MSCs cultured on collagen I and IV reduced expression of SMC and MHC compared to plastic. On ACM-HA, BM-MSCs maintained multipotent state by differentiating to bone and fat when induced. In Mesencult, BM-MSC-seeded ACM-HA expressed α-SMA, calponin, and MHC. TGF-ß1 and PDGF-BB enhanced SMC differentiation on collagens and ACM-HA. SMC proteins expression by BM-MSC varies depending on culture substrate. SMC markers are expressed higher on plastic and lower on collagen I, IV, and ACM-HA, suggesting these substrates preferentially maintain undifferentiated state of BM-MSC, which could be advantageous for incorporation of cell-seeded grafts to permit host modulation of tissue regeneration.

Bladder tissue engineering: tissue regeneration and neovascularization of HA-VEGF-incorporated bladder acellular constructs in mouse and porcine animal models.

Successful tissue engineering requires appropriate recellularization and vascularization. Herein, we assessed the regenerative and angiogenic effects of porcine bladder acellular matrix (ACM) incorporated with hyaluronic acid (HA) and vascular endothelial growth factor (VEGF) in mouse and porcine models. Prepared HA-ACMs were rehydrated in different concentrations of VEGF (1, 2, 3, 10, and 50 ng/g ACM). Grafts were implanted in mice peritoneum in situ for 1 week. Angiogenesis was quantified with CD31 and Factor VIII immunostaining using Simple PCI. Selected optimal VEGF concentration that induced maximum vascularization was then used in porcine bladder augmentation model. Implants were left in for 4 and 10 weeks. Three groups of six pigs each were implanted with ACM alone, HA-ACM, and HA-VEGF-ACM. Histological, immunohistochemical (Uroplakin III, alpha-SMA, Factor VIII), and immunofluorescence (CD31) analysis were performed to assess graft regenerative capacity and angiogenesis. In mouse model, statistically significant increase in microvascular density was demonstrated in the 2 ng/g ACM group. When this concentration was used in porcine model, recellularization increased significantly from weeks 4 to 10 in HA-VEGF-ACM, with progressive decrease in fibrosis. Significantly increased vascularization, coupled with increased urothelium and smooth muscle cell (SMC) regeneration, was observed in HA-VEGF grafts at week 10 in the center and periphery, compared with week 4. HA-VEGF grafts displayed highest in vivo epithelialization, neovascularization, and SMCs regeneration. A total of 2 ng/g tissue VEGF when incorporated with HA proved effective in stimulating robust graft recellularization and vascularization, coordinated with increased urothelial bladder development and SMC augmentation into bundles by week 10.

Urinary bladder tissue engineering using natural scaffolds in a porcine model: role of Toll-like receptors and impact of biomimetic molecules.

INTRODUCTION: Natural scaffolds have been shown to induce T helper 2 (TH2)-specific immune responses in host tissues; however, the precise mechanisms that underlie this immune response are unknown. Using a porcine animal model, we evaluated the role of Toll-like receptors (TLRs) and matrix remodelling in the implantation of bladder acellular matrix (ACM) grafts and ACMs fortified with biomimetic materials. MATERIALS AND METHODS: Bladders were decellularized with detergent and treated in 3 different ways prior to implantation: ACM alone, hyaluronic acid (HA)-ACM and HA-vascular endothelial growth factor (VEGF)-ACM. Animals were sacrificed at 4 or 10 weeks post-implantation and total gene expressions for TH2 (IL-4), TH1 (IFN-γ), TLR2, TLR4, and TGF-ß1 were analyzed using real-time RT-PCR. Using histology (H&E and Masson's trichrome) and immunohistochemistry (uroplakin, α-smooth muscle actin, CD31 and factor VIII) the regenerative capacity was correlated with the gene expression of different proteins. RESULTS: IL-4, TLR2, and TLR4 gene expression were markedly decreased at 4 and 10 weeks in both the HA-ACM group and the HA-VEGF-ACM group compared to ACM alone. IFN-γ expression was negligible in all groups and time periods. TGF-ß1 expression was highest in the HA- and VEGF-treated grafts. Recellularization was inversely proportional to TLR and TH2 expression but proportional to TGF-ß1. CONCLUSION: ACM alone grafts demonstrated stronger TLR4 expression which may promote a distinct TH2 immune response and a reduced regenerative capacity in grafts. Treatment of grafts with HA and VEGF may help regulate host immune responses by reducing TLR4 and IL-4 and increasing TGF-ß1.

Quantitative magnetic resonance imaging assessment of matrix development in cell-seeded natural urinary bladder smooth muscle tissue-engineered constructs.

The approach of cell-seeded natural scaffolds holds great promise for tissue engineering complicated soft-tissue organs such as the urinary bladder and heart. However, relatively little is known about cell-natural scaffold interactions or their influence on magnetic resonance imaging (MRI) characterization, which is valuable for noninvasive monitoring. Ideally, MRI should provide information on tissue biochemistry in addition to structure and function. In this study, quantitative MRI was performed on control and smooth muscle cell-seeded natural bladder matrices at different time points up to 7 days postseeding. Measurements of MR relaxation times (T1 and T2) and diffusion coefficient (D) showed an overall change that was incompatible with cell presence. Multicomponent T2 provided greater specificity, revealing time-course changes in the short T2 fraction that were consistent with biochemically determined matrix degradation from collagenase released from seeded cells. These matrix alterations are noted for the first time, and their relatively early occurrence may be unique to soft-tissue matrices compared with synthetic materials. More importantly, they are not evident on histology but are revealed on quantitative MRI. We conclude that quantitative MRI may provide specific information on cell-matrix interaction and is a promising noninvasive approach to understand and monitor cell-seeded natural scaffold-based regeneration.

Squalene selectively protects mouse bone marrow progenitors against cisplatin and carboplatin-induced cytotoxicity in vivo without protecting tumor growth.

Squalene, an isoprenoid antioxidant is a potential cytoprotective agent against chemotherapy-induced toxicity. We have previously published that squalene protects light-density bone marrow cells against cis-diamminedichloroplatinum( II) (cisplatin)-induced toxicity without protecting tumor cells in vitro. Here, we developed an in vivo mouse model of cisplatin and cis-diammine (cyclobutane-1,1-dicarboxylato) platinum(II) (carboplatin)-induced toxicity to further investigate squalene-mediated LD-BM cytoprotection including the molecular mechanism behind selective cytoprotection. We found that squalene significantly reduced the body weight loss of cisplatin and carboplatin-treated mice. Light-density bone marrow cells from squalene-treated mice exhibited improved formation of hematopoietic colonies (colony-forming unit-granulocyte macrophage). Furthermore, squalene also protected mesenchymal stem cell colonies (colony-forming unit-fibroblast) from cisplatin and carboplatin-induced toxicity. Squalene-induced protection was associated with decreased reactive oxygen species and increased levels of glutathione and glutathione peroxidase/glutathione-S-transferase. Importantly, squalene did not protect neuroblastoma, small cell carcinoma, or medulloblastoma xenografts against cisplatin-induced toxicity. These results suggest that squalene is a potential candidate for future development as a cytoprotective agent against chemotherapeutic toxicity.

Prospective validation of the pediatric appendicitis score.

OBJECTIVE: To prospectively validate the Pediatric Appendicitis Score (PAS), developed on a cohort of children with abdominal pain suggestive of appendicitis, in unselected children with abdominal pain who present to the emergency department. STUDY DESIGN: Over a 19-month period, we prospectively recruited children 1 to 17 years old who came to our tertiary pediatric emergency department, with a chief complaint of abdominal pain of duration less than 7 days. PAS components included fever >38 degrees C, anorexia, nausea/vomiting, cough/percussion/hopping tenderness (2 points), right-lower-quadrant tenderness (2 points), migration of pain, leukocytosis >10 000 cells/mm(3), and polymorphonuclear neutrophilia > 7500 cells/mm(3). A follow-up call was made to verify final outcome. Sensitivity, specificity, and the receiver operating characteristic curve of the PAS with respect to diagnosis of appendicitis were calculated. RESULTS: We collected data on 849 children. 123 (14.5%) had pathologic study-proven appendicitis. Mean (median, range) score for children with appendicitis and without appendicitis was 7.0 (7, 2-10) and 1.9 (1, 0-9), respectively. If a cutoff PAS of or=7 was used to take children to the operating room without further investigation, only 29 (4%) would not have appendicitis. For the PAS the area under the receiver operator curve was 0.95. CONCLUSIONS: The PAS is useful, because a value or=7 (found in 61% of children with appendicitis) has a high validity for predicting the presence of appendicitis. Children with PAS of 3 to 6 (37% with appendicitis and 23% without appendicitis in this study) should undergo further investigation such as observation, ultrasonography, or computed tomography.

Porcine bladder acellular matrix (ACM): protein expression, mechanical properties.

Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization.

Feasibility study of a novel urinary bladder bioreactor.

We have devised a bioreactor to simulate normal urinary bladder dynamics. The design permits a cell-seeded scaffold made from a modified porcine acellular matrix to be placed between 2 closed chambers filled with culture medium and be mechanically stimulated in a physiologically relevant manner. Specifically designed software increased hydrostatic pressure from 0 to 10 cm of water in a linear fashion in 1 chamber, resulting in mechanical stretch and strain on the scaffold. Pressure was increased over 55 min (filling) and then decreased to 0 over 10 s (voiding). Commercially available small intestinal submucosa scaffolds were used to test the mechanical capabilities of the bioreactor, and pressure waveforms were generated for up to 18 h. Scaffolds were seeded with bladder smooth muscle or urothelial cells and incubated in the bioreactor, which generated pressure waveforms for 6 h. Scaffold integrity was preserved as seen through Masson's trichrome staining. No obvious contamination of the system was noted. Hematoxylin and eosin staining showed presence of cells after incubation in the bioreactor, and immunohistochemistry and real-time reverse transcriptase polymerase chain reaction suggested continued cellular activity. Cellular orientation tended to be perpendicular to the applied pressure. Preliminary results suggest that our bioreactor is a suitable model for simulating normal physiological conditions of bladder cycling in an ex vivo system.

Culture results via the internet: a novel way for communication after an emergency department visit.

OBJECTIVE: To determine whether the Internet could be used to facilitate personal delivery of culture results to care givers after patient discharge from the pediatric emergency department. STUDY DESIGN: We recruited families of children who had cultures taken and were discharged home from our tertiary pediatric emergency department. Parents were given a unique ID and password to retrieve information on culture results from the study web-site. Results were posted and an e-mail was sent to the family. Access pattern to the web-site was recorded, and follow-up calls at 5 and 10 days after posting were made. RESULTS: A total of 527 families were approached; 224 were excluded. Of 303 cultures available, 24 (8%) were positive and 5 (2%) were considered to be contaminants. 186 (61%) parents accessed the Internet-system after mean 94 hours (range 1 minute to 611 hours) after posting. Of the 243 (80%) families reached for follow-up, 66 (27%) "had no time" to enter the website. CONCLUSIONS: This web-based follow-up system is valuable for negative cultures but access by parents is delayed for positive cultures. Future effort to increase awareness regarding importance of obtaining culture results is needed.