Expression of Matrix Metalloproteinases in the Circulating Immune Cells in Children with Helicobacter pylori Infection-Correlation with Clinical Factors.

H. pylori gastritis is strongly associated with the upregulation of the expression of several matrix metalloproteinases (MMPs) in the gastric mucosa. However, the role of MMP-2 and MMP-9, and their inhibitors (tissue inhibitors of metalloproteinases -TIMPs) produced by immune cells in infected children have not been clearly defined. Moreover, the effects of H. pylori eradication therapy on MMPs and TIMPs production has not been evaluated. A total of 84 children were studied: 24-with newly diagnosed H. pylori gastritis, 25-after H. pylori eradication therapy (17 of them after successful therapy), 24-with H. pylori-negative gastritis, and 11-controls. Plasma levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 by ELISA; MMPs and TIMPs expression in lymphocytes; neutrophils and monocytes in peripheral blood by multiparameter flow cytometry; and mucosal mRNA expression levels of MMPs and TIMP-1 in gastric biopsies by RT-PCR were evaluated. Children with H. pylori-related gastritis showed the following: (1) increased MMP-2 and TIMP-2 plasma levels, (2) increased intracellular expression of MMP-2 in the circulating lymphocytes and neutrophils, (3) low frequencies of circulating TIMP-1+ and TIMP-2+ leukocytes, and (4) high expression of mRNA for MMP-9 along with low expression of mRNA for MMP-2 in the gastric mucosa. Unsuccessful H. pylori eradication was associated with the following: (1) high plasma levels of MMP-9 and TIMP-1, (2) increased pool of TIMP-1+ lymphocytes as well as high expression of MMP-9 in circulating lymphocytes, and (3) high expression of mRNA for MMP-9 in the gastric mucosa. Our data suggest that MMPs are important contributors to stomach remodelling in children with H. pylori-related gastritis. Unsuccessful H. pylori eradication is associated with increased MMP-9 in plasma, circulating lymphocytes, and gastric mucosa.

Effect of four fluoroquinolones on the viability of bladder cancer cells in 2D and 3D cultures.

Introduction: The anticancer properties of fluoroquinolones and the high concentrations they achieve in urine may help in bladder cancer therapy. This study aimed to analyze the properties of 4 fluoroquinolones as potential candidates for supportive treatment of bladder cancer. Methods: Comparative analyses were performed on the cytotoxic effects of norfloxacin, enrofloxacin, moxifloxacin, and ofloxacin on normal and cancer urothelial cell lines. In 2D culture, the cytotoxic properties of fluoroquinolones were evaluated using MTT assay, real-time cell growth analysis, fluorescence and light microscopy, flow cytometry, and molecular analysis. In 3D culture, the properties of fluoroquinolones were tested using luminescence assays and confocal microscopy. Results and Discussion: All tested fluoroquinolones in 2D culture decreased the viability of both tested cell lines in a dose- and timedependent manner. Lower concentrations did not influence cell morphology and cytoskeletal organization. In higher concentrations, destruction of the actin cytoskeleton and shrinkage of the nucleus was visible. Flow cytometry analysis showed cell cycle inhibition of bladder cancer cell lines in the G2/M phase. This influence was minimal in the case of normal urothelium cells. In both tested cell lines, increases in the number of late apoptotic cells were observed. Molecular analysis showed variable expression of studied genes depending on the drug and concentration. In 3D culture, tested drugs were effective only in the highest tested concentrations which was accompanied by caspase 3/7 activation and cytoskeleton degradation. This effect was hardly visible in non-cancer cell lines. According to the data, norfloxacin and enrofloxacin had the most promising properties. These two fluoroquinolones exhibited the highest cytotoxic properties against both tested cell lines. In the case of norfloxacin, almost all calculated LC values for bladder cancer cell lines were achievable in the urine. Enrofloxacin and norfloxacin can be used to support chemotherapy in bladder cancer patients.

Urinary bladder augmentation with acellular biologic scaffold-A preclinical study in a large animal model.

Current strategies in urinary bladder augmentation include use of gastrointestinal segments, however, the technique is associated with inevitable complications. An acellular biologic scaffold seems to be a promising option for urinary bladder augmentation. The aim of this study was to evaluate the utility of bladder acellular matrix (BAM) for reconstruction of clinically significant large urinary bladder wall defects in a long-term porcine model. Urinary bladders were harvested from 10 pig donors. Biological scaffolds were prepared by chemically removing all cellular components from urinary bladder tissue. A total of 10 female pigs underwent hemicystectomy and subsequent bladder reconstruction with BAM. The follow-up study was 6 months. Reconstructed bladders were subjected to radiological, macroscopic, histological, immunohistochemical, and molecular evaluations. Six out of ten animals survived the 6-month follow-up period. Four pigs died during observation due to mechanical failure of the scaffold, anastomotic dehiscence between the scaffold and native bladder tissue, or occluded catheter. Tissue engineered bladder function was normal without any signs of postvoid residual urine in the bladder or upper urinary tracts. Macroscopically, graft shrinkage was observed. Urothelium completely covered the luminal surface of the graft. Smooth muscle regeneration was observed mainly in the peripheral graft region and gradually decreased toward the center of the graft. Expression of urothelial, smooth muscle, blood vessel, and nerve markers were lower in the reconstructed bladder wall compared to the native bladder. BAM seems to be a promising biomaterial for reconstruction of large urinary bladder wall defects. Further research on cell-seeded BAM to enhance urinary bladder regeneration is required.

A tissue-engineered urinary conduit in a porcine urinary diversion model.

The use of an ileal segment is a standard method for urinary diversion after radical cystectomy. Unfortunately, utilization of this method can lead to numerous surgical and metabolic complications. This study aimed to assess the tissue-engineered artificial conduit for urinary diversion in a porcine model. Tissue-engineered tubular polypropylene mesh scaffolds were used for the right ureter incontinent urostomy model. Eighteen male pigs were divided into three equal groups: Group 1 (control ureterocutaneostomy), Group 2 (the right ureter-artificial conduit-skin anastomoses), and Group 3 (4 weeks before urostomy reconstruction, the artificial conduit was implanted between abdomen muscles). Follow-up was 6 months. Computed tomography, ultrasound examination, and pyelogram were used to confirm the patency of created diversions. Morphological and histological analyses were used to evaluate the tissue-engineered urinary diversion. All animals survived the experimental procedures and follow-up. The longest average patency was observed in the 3rd Group (15.8 weeks) compared to the 2nd Group (10 weeks) and the 1st Group (5.8 weeks). The implant's remnants created a retroperitoneal post-inflammation tunnel confirmed by computed tomography and histological evaluation, which constitutes urostomy. The simultaneous urinary diversion using a tissue-engineered scaffold connected directly with the skin is inappropriate for clinical application.

Molecular Aspects of Adipose-Derived Stromal Cell Senescence in a Long-Term Culture: A Potential Role of Inflammatory Pathways.

Long-term culture of mesenchymal stromal/stem cells in vitro leads to their senescence. It is very important to define the maximal passage to which the mesenchymal stromal/stem cells maintain their regenerative properties and can be used for cellular therapies and construction of neo-organs for clinical application. Adipose-derived stromal/stem cells were isolated from porcine adipose tissue. Immunophenotype, population doubling time, viability using bromodeoxyuridine assay, MTT assay, clonogencity, ß-galactosidase activity, specific senescence-associated gene expression, apoptosis, and cell cycle of adipose-derived mesenchymal stromal/stem cells (AD-MSCs) were analyzed. All analyses were performed through 12 passages (P). Decreasing viability and proliferative potential of AD-MSCs with subsequent passages together with prolonged population doubling time were observed. Expression of ß-galactosidase gradually increased after P6. Differentiation potential of AD-MSCs into adipogenic, chondrogenic, and osteogenic lineages decreased at the end of culture (P10). No changes in the cell cycle, the number of apoptotic cells and expression of specific AD-MSC markers during the long-term culture were revealed. Molecular analysis showed increased expression of genes involved in activation of inflammatory response. AD-MSCs can be cultured for in vivo applications without loss of their properties up to P6.

CD133 Antigen as a Potential Marker of Melanoma Stem Cells: In Vitro and In Vivo Studies.

Melanoma is the most dangerous type of skin cancer. Cancer stem cells (CSCs) are suspected to be responsible for the cancer recurrence and in the consequence for cancer therapy failure. CD133 is a potential marker for detection of melanoma CSCs. Experiments were performed on the B16-F10 mouse melanoma cell line. CD133+ cells were isolated using an immunomagnetic cell sorting technique. After isolation proliferative and clonogenic potential of CD133+, CD133- and CD133+/- were evaluated. The potential of CD133+ and CD133- cells for tumor induction was conducted on C57BL/6J mouse model. Three different cell quantities (100, 1000, 10000) were tested. Tumor morphology, number of mitoses, and tumor necrosis area were analyzed. Average 0.12% CD133+ cells were isolated. Compared to CD133- and unsorted CD133+/- cells, CD133+ cells were characterized by the higher proliferative and clonogenic potential. These properties were not confirmed in vivo, as both CD133+ and CD133- cells induced tumor growth in mouse model. No statistical differences in mitosis number and tumor necrosis area were observed. Simultaneous detection of CD133 antigen with other markers is necessary for accurate identification of these melanoma cancer stem cells.

Mesenchymal stromal cells modulate the molecular pattern of healing process in tissue-engineered urinary bladder: the microarray data.

BACKGROUND: Molecular mechanisms underlying the regenerative process induced by stem cells in tissue-engineered urinary bladder are poorly explained. The study was performed to explore the pathways associated with regeneration process in the urinary bladder reconstructed with adipose tissue-derived mesenchymal stromal cells (ASCs). METHODS: Rat urinary bladders were reconstructed with bladder acellular matrix (BAM) (n = 52) or BAM seeded with adipose tissue-derived mesenchymal stromal cells (ASCs) (n = 52). The process of bladder healing was analyzed at 7, 30, 90, and 180 days postoperatively using macroscopic histologic and molecular techniques. Gene expression was analyzed by microarrays and confirmed by real-time PCR. RESULTS: Numerous differentially expressed genes (DEGs) were identified between the bladders augmented with BAM seeded with ASCs or BAM only. Pathway analysis of DEGs allows to discover numerous pathways among them Hedgehog, TGF-ß, Jak-STAT, PI3-Akt, and Hippo modulated by ASCs during the healing process of tissue-engineered urinary bladder. Real-time PCR analysis confirmed upregulation of genes involved in the Hedgehog signaling pathway including Shh, Gli1, Smo, Bmp2, Bmp4, Wnt2, Wnt2b, Wnt4, Wnt5a, and Wnt10 in urinary bladders reconstructed with ASC-seeded grafts. CONCLUSION: The study provided the unequivocal evidence that ASCs change the molecular pattern of healing in tissue-engineered urinary bladder and indicated which signaling pathways triggered by ASCs can be associated with the regenerative process. These pathways can be used as targets in the future studies on induced urinary bladder regeneration. Of particular interest is the Hedgehog signaling pathway that has been upregulated by ASCs during healing of tissue-engineered urinary bladder.

The different expression of key markers on urothelial holoclonal, meroclonal, and paraclonal cells in in vitro culture.

Urothelial cell populations which differ in morphology and proliferation capacities can be isolated from the urinary bladder. The goal of this study was to analyze a clonal, proliferative, and self-renewing potential of porcine urothelial cells and to compare expression of selected adhesion and tight junction molecules, urothelial and stem cell markers for the urothelial clone types. Urothelial cells were isolated from 10 porcine urinary bladders. Three different clone types: holoclone-, meroclone-and paraclone-like colonies were identified based on their morphology. To characterize and compare the urothelial clones the immunofluorescent stains were performed. Expression of pancytokeratin (PanCK), Ki-67 and p63 was higher for holoclone- like cells compared to meroclone-and paraclone-like cells (P < 0.05). Meroclone-like cells expressed higher levels of p63 compared to paraclone- like cells (P < 0.05). The level of Ki-67 and PanCK for meroclone- and paraclone- like cells was comparable (P > 0.05). ß1 and ß4 integrins were not expressed. Expression of zonula occludens-1 (ZO-1) in cell-cell junctions for paraclone-, meroclone-and holoclone-like cells was 17.6 ± 0.6, 14.7 ± 0.5, and 16.1 ± 0.4, respectively. The results of actin filaments (F-actin) expression were 253,634 ± 6,920 for meroclone-like cells, 198,512 ± 7,977 for paraclone-like cells and 133,544 ± 3,169 for holoclone-like cells. Three urothelial cell types with differing features can be isolated from the bladder. Holoclone-like cells are the richest in stem cells and should be used in further studies for construction of neo-bladder or neo-conduit using tissue engineering methods.

Understanding the role of mesenchymal stem cells in urinary bladder regeneration-a preclinical study on a porcine model.

BACKGROUND: The tissue engineering of urinary bladder advances rapidly reflecting clinical need for a new kind of therapeutic solution for patients requiring urinary bladder replacement. Majority of the bladder augmentation studies have been performed in small rodent or rabbit models. Insufficient number of studies examining regenerative capacity of tissue-engineered graft in urinary bladder augmentation in a large animal model does not allow for successful translation of this technology to the clinical setting. The aim of this study was to evaluate the role of adipose-derived stem cells (ADSCs) in regeneration of clinically significant urinary bladder wall defect in a large animal model. METHODS: ADSCs isolated from a superficial abdominal Camper's fascia were labeled with PKH-26 tracking dye and subsequently seeded into bladder acellular matrix (BAM) grafts. Pigs underwent hemicystectomy followed by augmentation cystoplasty with BAM only (n = 10) or BAM seeded with autologous ADSCs (n = 10). Reconstructed bladders were subjected to macroscopic, histological, immunofluoresence, molecular, and radiological evaluations at 3 months post-augmentation. RESULTS: Sixteen animals (n = 8 for each group) survived the 3-month follow-up without serious complications. Tissue-engineered bladder function was normal without any signs of post-voiding urine residual in bladders and in the upper urinary tracts. ADSCs enhanced regeneration of tissue-engineered urinary bladder but the process was incomplete in the central graft region. Only a small percentage of implanted ADSCs survived and differentiated into smooth muscle and endothelial cells. CONCLUSIONS: The data demonstrate that ADSCs support regeneration of large defects of the urinary bladder wall but the process is incomplete in the central graft region. Stem cells enhance urinary bladder regeneration indirectly through paracrine effect.

Stem cells and differentiated cells differ in their sensitivity to urine in vitro.

Urinary tract regeneration using tissue engineering is one of the most challenging issues in the field of reconstructive urology. Cells seeded on scaffold are exposed to urine immediately after the implantation. The outcome of urinary bladder regeneration is depended on the ability of these cells to survive, proliferate, and regenerate. The aim of this study was to compare a sensitivity of three different cell lines to urine in vitro. Three different cell lines were isolated from porcine bladder (urothelial cells, UCs and smooth muscle cells, SMCs) and adipose tissue (adipose-derived stem cells, ADSCs). Cell viability (MTT assay), proliferation (real-time cell analysis using xCELLigence system) and apoptosis/necrosis (flow cytometry) were analyzed after exposition to urine. ADSCs were the most sensitive to urine compared to two other tested cell lines. Among the bladder cell lines the UCs were more resistant to urine than SMCs. Twenty four hour incubation of UCs, SMCs, and ADSCs with urine lead to ∼40%, ∼70%, and ∼90% reduction of their viability, respectively. The mechanism of urine mediated cytotoxicity differed depending on the tested cell type. Urothelial and SMCs seems to be more suitable for urinary bladder regeneration compared to mesenchymal stem cells, however, these cells have limited application especially in the case of urinary bladder cancer.

Isolation, expansion and characterization of porcine urinary bladder smooth muscle cells for tissue engineering.

BACKGROUND: A key requirements for therapy utilizing the tissue engineering methodologies is use of techniques which have the capability to yield a high number of cells, from small tissue biopsy in a relatively short time. Up to date there was no optimal methods of isolation and expansion of urinary bladder smooth muscle cells (UB-SMCs). The aim of this study was to compare isolation and expansion techniques of UB-SMCs to select the most repeatable and efficient one. METHOD: Five protocols of porcine UB- SMCs isolation including enzymatic and explant techniques and three expansion techniques were compared. Isolation effectiveness was evaluated using trypan blue assay. Cell phenotype was confirmed by immunofluorescence staining. Proliferation rate was analyzed using MTT and X- Celligence system. Cellular senescence was assessed measuring ß-galactosidase activity. RESULTS: Enzymatic methods using collagenase with dispase (method I) or collagenase only (method III) allowed to isolate much larger number of cells than the methods using trypsin with collagenase (method II) and collagenase after digestion with trypsin (method IV). The success rate of establishment of primary culture was the highest when the isolated cells were cultured in the Smooth muscle Growth Medium-2 (SmGM-2). Expression of the smooth muscle markers- alpha smooth muscle actin and smoothelin was the highest for cells isolated by enzymatic method I and cultured in SmGM-2. There was no significant signs of cell senescence until the 8th passage. CONCLUSION: The most efficient method of establishment of porcine UB-SMCs culture is enzymatic digestion of urinary bladder tissue with collagenase and dispase and culture of isolated cells in SmGM-2. This method was up to 10 times more efficient than other methods used for isolation and culture of UB-SMCs. This is an easy and consistent method for obtaining high numbers of urinary bladder smooth muscle cells.

Optimization of porcine urothelial cell cultures: Best practices, recommendations, and threats.

Many experimental approaches have been conducted in order to isolate urothelial cells from bladder tissue biopsies, but each method described has utilized different protocols and sources of bladder tissue. In this study, we compared the different methods of urothelial cell isolation available in literature together with standardized methods in order to obtain more unified results. Five methods for primary porcine urothelial culture establishment were compared: tissue explants and four enzymatic methods utilizing collagenase II, dispase II, combination of dispase II and trypsin, and trypsin alone. The average number of isolated cells, cell morphology, success of established culture, average number of cells from the first passage, expression of p63 and pancytokeratin and the characterization of urothelial cell growth, and aging were analyzed during the in vitro culture. The method utilizing dispase II was the most efficient and reproducible method for the isolation and culture of porcine urothelial cells when compared to the other tested methods. Urothelial cells obtained by this method grew considerably well and the cultures were established with high efficiency, which enabled us in obtaining a large quantity of cells with normal morphology. Contamination with fibroblasts in this method was the lowest. The utilization of a proper method for urothelial cell isolation is a critical step in the urinary tract regeneration when using tissue engineering techniques. In summary, this study demonstrated that by utilizing the described method with dispase II, a suitable number of cells was achieved, proving the method useful for tissue regeneration.

Targeted therapy for stress urinary incontinence: a systematic review based on clinical trials.

INTRODUCTION: Controversy exists regarding the therapeutic benefit of cell-based therapy in the treatment of stress urinary incontinence (SUI). AREAS COVERED: The aim of this systematic review was to evaluate evidence regarding the therapeutic effect and safety of cell-based therapy in the treatment of SUI and to propose a new approach to SUI treatment utilizing tissue engineering methodologies. We have thoroughly reviewed the literature using PubMed in order to identify only original, clinical studies involving cell therapy for SUI. EXPERT OPINION: Cell-based therapy, as practiced today, is a safe but ineffective method for SUI treatment. The key to an optimal therapeutic outcome in SUI is accurate diagnosis combined with targeted therapy. Targeted therapy in SUI should be based on cell implantation to restore and regenerate the damaged urethral sphincter and/or the construction of a neo-pubourethral ligament utilizing tissue engineering methodologies.