Mumio (Shilajit) as a potential chemotherapeutic for the urinary bladder cancer treatment.

Mumio (Shilajit) is a traditional medicinal drug known and used for hundreds of years. Bladder cancer is one of the most common cancer types and better treatments are needed. This study analysed the in vitro effect of Mumio on urinary bladder cancer cells (T24 and 5637) in comparison to normal uroepithelial cells (SV-HUC1). Cytotoxicity of Mumio was analysed in these cell lines via MTT and real-time cell growth assays as well via the assessment of the cytoskeleton, apoptosis, and cell cycle. Mumio affected the viability of both cell types in a time and concentration dependent manner. We observed a selectivity of Mumio against cancer cells. Cell cycle and apoptosis analysis showed that Mumio inhibited G0/G1 or S phase cell cycle, which in turn induced apoptosis. Our results showed that Mumio was significantly more cytotoxic to urinary bladder cancer cells than to normal cells. These results are promising and indicate Mumio as a great candidate for urinary bladder cancer treatment and further investigations should be performed.

The role of early diagnosis of emphysematous cystitis: A case report and literature review.

Emphysematous cystitis (EC) is a rare entity caused by bacteria, which produce gas filled cysts in the bladder wall. We present a case of EC in a 72-year-old woman admitted to Vascular Surgery Department because of diabetic foot syndrome. During the hospital stay, the patient's general condition deteriorated. CT established EC diagnosis. Surgical treatment was inevitable. Salvage cystectomy was performed. Despite macroscopic removal of necrotic tissues, the condition of the patient didn't improve, 75 days past diagnosis of EC she died due to the multi-organ failure. Prompt diagnosis provided by imaging plays a key role in the treatment of EC.

The development of marine biomaterial derived from decellularized squid mantle for potential application as tissue engineered urinary conduit.

Tissue engineering is focusing research effort on search for new biomaterials that might be applied to create artificial urinary conduit. Nevertheless, the demanding biomechanical characteristics necessary for proper conduit function is difficult to be replicated. In this study, we are introducing novel marine biomaterial obtained by decellularization of squid mantle derived from Loligo vulgaris. Squid mantles underwent decellularization according to developed dynamic flow two-staged procedure. Efficacy of the method was confirmed by computational dynamic flow analysis. Subsequently Decellularized Squid Mantle (DSM) underwent extensive histological analysis and mechanical evaluation. Based on gained biomechanical data the computational modelling using finite element method was utilized to simulate behavior of DSM used as a urinary conduit. Taking into account potential application in reconstructive urology, the DSM was then evaluated as a scaffold for urothelial and smooth muscle cells derived from porcine urinary bladder. Conducted analysis showed that DSM created favorable environment for cells growth. In addition, due to polarized structure and natural external polysaccharide layer, it protected seeded cells from urine.

Development of a conductive biocomposite combining graphene and amniotic membrane for replacement of the neuronal network of tissue-engineered urinary bladder.

Tissue engineering allows to combine biomaterials and seeded cells to experimentally replace urinary bladder wall. The normal bladder wall however, includes branched neuronal network propagating signals which regulate urine storage and voiding. In this study we introduced a novel biocomposite built from amniotic membrane (Am) and graphene which created interface between cells and external stimuli replacing neuronal network. Graphene layers were transferred without modifying Am surface. Applied method allowed to preserve the unique bioactive characteristic of Am. Tissue engineered constructs composed from biocomposite seeded with smooth muscle cells (SMC) derived from porcine detrusor and porcine urothelial cells (UC) were used to evaluate properties of developed biomaterial. The presence of graphene layer significantly increased electrical conductivity of biocomposite. UCs and SMCs showed an organized growth pattern on graphene covered surfaces. Electrical filed stimulation (EFS) applied in vitro led additionally to increased SMCs growth and linear arrangement. 3D printed chamber equipped with 3D printed graphene based electrodes was fabricated to deliver EFS and record pressure changes caused by contracting SMCs seeded biocomposite. Observed contractile response indicated on effective SMCs stimulation mediated by graphene layer which constituted efficient cell to biomaterial interface.

Rare neoplasm of genito-urinary tract. Genito-urinary sarcomas: chemotherapy.

PURPOSE OF REVIEW: The purpose of this article was to describe the systemic therapy of genito-urinary sarcomas. RECENT FINDINGS: High rate of distant metastasis and high mortality rate has brought interest into the development of new therapeutic approaches. Various modules of chemotherapy were sampled in sarcoma treatment, although clinical response is still unsatisfactory. Chemotherapy in sarcomas can be used as neoadjuvant or adjuvant to the surgery. There is no consensus on the current role of adjuvant chemotherapy. Study results are conflicting; therefore, conclusions drawn from the studies are uncertain. In general, the adjuvant chemotherapy is not standard treatment in adult-type sarcomas. In addition, chemotherapy for advanced and metastatic sarcoma disease, as well as second-line chemotherapy, was discussed. SUMMARY: The best treatment for sarcomas in case of organ-confined disease and in selected cases of locally advanced disease seems to be surgery followed by chemotherapy. In case of metastasis stage of sarcoma, preoperative chemotherapy with surgery of residual masses should be considered as first-line treatment followed by postoperative chemotherapy. The treatment of patient with highly advanced disease and/or unresectable metastases should be individualized (chemotherapy, radiotherapy and best supportive care).

Urine--a waste or the future of regenerative medicine?

In recent years, urine has emerged as a source of urine cells. Two different types of cells can be isolated from urine: urine derived stem cells (USCs) and renal tubular cells called urine cells (UCs). USCs have great differentiation properties and can be potentially used in genitourinary tract regeneration. Within this paper, we attempt to demonstrate that such as easily accessible source of cells, collected during completely non-invasive procedures, can be better utilized. Cells derived from urine can be isolated, stored, and used for the creation of urine stem cell banks. In the future, urine holds great potential to become a main source of cells for tissue engineering and regenerative medicine.

New application of carbon nanotubes in haemostatic dressing filled with anticancer substance.

The drug-carrier system used as innovative haemostatic dressing with oncostatic action is studied. It is obtained from CDDP (cisplatin) doped SWCNT (single walled carbon nanotubes), modified and purified by H2O2 in hydrothermal treatment process. In the in vivo nephron sparing surgery (NSS) study we used 35 BALB/c nude mice with induced renal cancer using adenocarcinoma 786-o cells. Animals were divided into four groups: CDDP(M-), CDDP(M+), CONTROL(M-) and CONTROL(M+). In CDDP(M-) and CDDP(M+) groups we used, intraoperatively, carbon nanotubes filled with cisplatin (CDDP). In CONTROL(M-) and CONTROL(M+) groups carbon nanotubes were used alone. During NSS free margin (M-) or positive margin (M+) was performed. In the CDDP(M-) group, we do not observe local tumor recurrences. In Group CDDP(M+) only one animal was diagnosed with tumor recurrence. In control groups the recurrent tumor formation was observed. In our study, it is shown that CDDP filled SWCNT inhibit cancer recurrence in animal model NSS study, and can be successfully applied as haemostatic dressings for local chemoprevention.

How to isolate urothelial cells? Comparison of four different methods and literature review.

The aim of this study is to present the comparison of four different methods for urothelial cell isolation and culture and compare them to methods cited in the literature. Four different techniques were examined for urothelium isolation from rat bladders. Isolation effectiveness was calculated using trypan blue assay. Confirmation of isolated cell phenotype and comparison with native bladder tissue was confirmed using immunohistochemical (IHC), immunocytochemical (ICC) and immunofluorescence (IF) analysis. The method with bladder inversion and collagenase P digestion resulted in the highest number of isolated cells. These cells showed positive expression of cytokeratin 7, 8, 18, α6-integrin and p63. Our results and the literature review showed that the best method for urothelium bladder isolation is dissection of the epithelium layer from other bladder parts and digestion of mechanically prepared tissue in a collagenase solution.

Morphological and urodynamic evaluation of urinary bladder wall regeneration: muscles guarantee contraction but not proper function--a rat model research study.

BACKGROUND: Numerous studies are ungoing to develop a substitute for the native urinary bladder wall. The principals of tissue engineering approaches to urinary bladder wall augmentation require a favorable environment for smooth muscle regeneration, which is crucial for bladder function. This study was performed to evaluate bone marrow mesenchymal stem cells (BMSC) seeded on to amniotic membranes fixed to Tachosil sponges as grafts for urinary bladder muscle layer augmentation in a syngenic rat model. MATERIALS AND METHODS: Amniotic membranes seeded with BMSC and covered by Tachosil sponges were implanted as multilayer grafts into nine rats to regenerate the urinary bladder wall. The control group consisted of 12 healthy rats. Urodynamic examinations included contraction, elasticity, compliance, and urinary bladder motor activity. Hematocylin and eosin and Masson's trichrome stains were used to evaluate muscle regeneration; histological data were digitally analyzed with the ImageJ tool. RESULTS: The area of muscle bundles ranged from 5% to 25% or 32% to 41% in control versus reconstructed bladders, respectively. Among nine animals with reconstructed urinary bladders, urodynamic evaluation revealed bladder motor hyperactivity with regular (n = 4) or irregular (n = 1) storage and voiding phases, as well as proper bladder motor activity with a large bladder capacity (n = 1). No bladder contractility was recorded in one case and large stones developed in two animals, which made functional studies impossible. CONCLUSIONS: Regenerated smooth muscle cells created an autonomic cell population that was poorly assimilated to the rest of the urinary bladder wall. The histological presence of a regenerated muscle layer did not guarantee proper urinary bladder function.

Urine is a highly cytotoxic agent: does it influence stem cell therapies in urology?

The state of art of stem cell therapies in urologic regenerative medicine is still under development. There are still many issues before advances in tissue engineering can be introduced for clinical application. The essential question is whether stem cells should be seeded on the urinary tract lumen side. The present experiment, using Real-Time Cell Analyzer (RTCA) DP (Dual Plate) of the xCellligence system (Roche Applied Science, Mannheim, Germany), allowed us to monitor cellular events in real time. In this study we examined the influence of urine on bone marrow-derived mesenchymal stem cells (MSC). Cells were exposed to medium mixed with urine (1:1), medium mix with PBS (Phosphate Buffered Saline) (1:1), only urine, and whole medium without cells as background. The cell number was significantly lower in all groups exposed on medium mixed with urine and urine alone. The results showed that urine is a highly cytotoxic agent whose role in urologic regenerative medicine is underestimated.

In vitro optimization of the Gallus domesticus oviduct epithelial cells culture.

The aim of this experiment was to establish an efficient method for isolation and further culture in vitro of the normal chicken oviduct epithelial cells (COEC) for cell-based research models. Different factors were tested to optimize COEC primary culture for repeatable results: the origin of isolated cells (oviduct Infundibulum or Magnum section); the oviduct tissue dissociation procedure (mechanical scrapping or mincing), tissue digestion times (15, 30 and 45 min), the culture plates coating (colagene I, polystyrene surface or 3T3 feeder layer), the growth media (classic DMEM/Ham's F12 and defined serum-free medium, Lonza Switzerland), incubation temperature (37 °C vs 41°C) and different cell seeding numbers: 0.2M, 0.5M and 1.0M cells/well. The COEC isolated by mincing the Infundibular neck and digestion of tissue for 30 min formed cell aggregates of bright colour and gave proliferating colonies of epithelial-like character which was the best result obtained from all applied procedures in our studies. The fibroblast-like cells considered as contaminants occurred only sporadically up to day 7 of culture. Seeding about 1M cells in 1 mL of serum-free medium onto 12-well dishes gave the optimal growth of colonies resulting in 5 to 7 confluent culture wells from a single oviduct sample. Feeder layer and collagen I did not improve adhesion of the COEC to the culture vessel. Adoption of 37 °C and 41 °C did not reveal apparent differences to the condition of cultured COEC. Cell differentiation and proliferation potential depends on number and replicative capacity of isolated progenitors. The progenitors are responsible for holoclones formation and good culture growth. The percentage of colonies developed from the cells isolated from Infundibulum was greater than that of other samples in our studies. We conclude that the model of COEC primary cultures from different segments of oviduct, in particular infundibulum, should be incorporated to the range of avian cells research as this work generates questions about undocumented sources of oviduct progenitor cells.

Ciprofloxacin as a prophylactic agent against prostate cancer: a "two hit" hypothesis.

More evidence indicate that prostate inflammation can lead to prostate cancer development. Prostate cancer affects elderly men. Prostate cancer prophylaxis is an important issue because life expectancy is very long now. Ciprofloxacin is an antibacterial agent used mainly in urinary tract infections and prostate inflammation. This drug acts also against cancer cells by the inhibition of topoisomerase II. These properties should allow it to inhibit the development of prostate cancer. Firstly, ciprofloxacin can stop the acute and chronic prostate inflammation which can lead to cancer development. Secondly, ciprofloxacin can potentially kill prostate cancer cells in their early stage of development. Ciprofloxacin accumulates mainly in the prostate after oral intake thus ciprofloxacin seems to be a perfect candidate as a prophylactic agent.

Prostate cancer which affects an elderly man is a feature of senescence (cellular) - a biology phenomenon.

UNLABELLED: Some prostate cancers are clinically significant (i.e. life-threatening) but others are not. Small proportion of elderly men dies of prostate cancer while most of them harbor tumor lesions in their prostates. The aim of this paper was to present late-life form of the prostate cancer, which differs from its aggressive counterpart that affects men between 55-65 years old and younger. The differences can be found in carcinogenesis risk factors, cancer biology and finally patients' survival. The most important is that these two clinical (age-related) forms of the prostate cancer are still undistinguishable in clinico-pathology reports and patients bearing different diseases are offered the same treatment. Potential mechanisms leading to development of the late-life clinically indolent prostate cancer are discussed. It seems that the key abnormalities are proteins involved in control of regenerative potential and cell senescence. CONCLUSIONS: We postulate that late-life low-grade (clinically indolent) prostate cancer subcategory should be established. This type of «cancer¼ should rather be viewed as a senescence-related feature and probably not treated at all.

Hair stem cells for bladder regeneration in rats: preliminary results.

BACKGROUND: A variety of tissue engineering techniques are currently under development or investigation for bladder augmentation, but so far no approach is clearly superior. The aim of this study was to compare the suitability for cystoplasty augmentation in rats of in vivo implanted acellular bladder matrices (BAM) previously seeded with hair follicle stem cells and that of matrices implanted without the cells. MATERIALS AND METHODS: The rat hair follicle stem cell line was positive for CD34, p63, and Ki-67. 1 x 10(6) cells from 34 to 40 passages seeded onto nine BAM scaffolds were cultured for one week. Nine other scaffolds were left unseeded. Scaffolds were grafted into a surgically created defect within the anterior bladder wall: nine rats with acellular matrices and nine with cell-seeded BAM. Rats observed for six months were killed in monthly intervals. We performed gross examination, X-ray cystography, and hematoxylin-eosin, cytokine (CK)-7, CK-20, myoglobin, and desmin staining of the excised bladders. RESULTS: Minimal adhesions were observed and urinary leakage was noted in one case. Two animals died in the acellular group. Rats developed stone disease in bladders reconstructed with acellular BAM. Bladder capacity was similar, but the shape was regular and characteristically oval only in bladders grafted with cell-seeded BAM. Muscle layers in the apical parts of the reconstructed bladder walls were extremely thin in the cases of acellular grafts and thicker in bladders reconstructed with cell-seeded grafts. Muscle layer regeneration was better in the cell-seeded group. Urothelium regenerated in all animals. CONCLUSIONS: We have shown that hair follicle stem cells may be used for rat bladder wall regeneration.

Primary cultures from rat vibrissae as a potential cell source for in vitro construction of urinary bladder wall grafts.

BACKGROUND: In vitro-constructed grafts can be used for human bladder augmentation. There are many diseases in which autologous cells cannot be used for this purpose. The aim of the present study was to examine the potential of rat vibrissae hair follicle cells to form cultures, which could serve as a source for in vitro creation of urinary bladder wall grafts. METHODS: Two hundred vibrissae were excised from young Wistar male rats. Two different digestions were performed, in dispase and in collagenase. All follicles were additionally incubated in trypsin and ethylenediamine tetraacetic acid. Two different culture media based on DMEM (Dulbecco's Modified Eagle's Medium) were used: the first was supplemented with keratinocyte growth factor (KGF) and the second with epidermal growth factor. Immunocytochemical detection of cytokeratin, CD34, p63, Ki-67 (proliferation index), and HMB45 (Human Melanoma Black 45) was performed. RESULTS: Forty-eight primary cultures of rat follicle vibrissae cells were established from 200 hair follicles (24% successful rate). Twenty-four primary cultures were obtained after dispase digestion and 24 after collagenase treatment. Each group was cultured in 2 different media. A heterogeneity of primary cultures was observed. Cells formed a monolayer within a period of 2 to 4 weeks. The 24 primary cultures established after dispase treatment exhibited monolayers of small cuboid cells expressing cytokeratin and CD34. In the 40th passage 20%-40% of cells expressed p63; 85% of these cells from late passages were positive for Ki-67, indicating preserved mitotic potential. Epithelial-like phenotype was observed after dispase digestion and cultivation in KGF-supplemented medium. After 3 weeks, the morphology of these cells changed into fibroblast-like. These cultures were negative for CD34. Fibroblast-like cell growth was observed after collagenase treatment in both KGF- and EGF-supplemented media. These cells were positive for the melanocyte cell marker (HMB45). CONCLUSIONS: Culture media and isolation conditions influence hair follicle stem cell differentiation. The stem cell niche within the hair follicles is a reservoir of cells, which can be potentially used for in vitro creation of urinary bladder wall grafts.

Influence of two Pt(IV) complexes on viability, apoptosis and cell cycle of B16 mouse melanoma tumors.

UNLABELLED: Several platinum(IV) complexes are showing considerable promise in initial trials, producing reactive intermediates that then interact with DNA. AIM: To perform in vitro study of two new platinum(IV) complexes cytotoxic effect on B16 mouse melanoma cells. METHODS: PtCl(4)(dbtp)(2) and PtCl(2)(6mp)(2) complexes were prepared. PtCl(4)(dbtp)(2) was created as modification of PtCl(4)(dmtp) test previously. Apoptosis and necrosis were examined using flow cytometry, upon Annexin V/PI staining. RESULTS: LC(10), LC(50) and LC(90) parameters established for PtCl(4)(dbtp)(2) were as following: 2.6, 17.0, 58.0 mumol/L. However LC(10) and LC(50) established for PtCl(2)(6mp)(2) were 1.2 and 14.0 micromol/l respectively. The both complexes induced apoptosis. PtCl(2)(6mp)(2) induced cell cycle arrest in G0/G1, while PtCl(4)(dbtp)(2) - in S-phase. CONCLUSIONS: PtCl(4)(dbtp)(2) appeared to be more cytotoxic against B16 cells than PtCl(2)(6mp)(2). Apoptosis was the main mechanism of cell loss in cultures incubated with both tested complexes.

Alginate is not a good material for growth of rapidly proliferating cells.

INTRODUCTION: Alginate scaffolds are widely used in tissue engineering. The aim of this study was to evaluate alginate as a scaffold for 3D cultures of rapidly proliferating cells. MATERIALS AND METHODS: Murine 3T3 fibroblasts were cultured in an alginate scaffold for 30 days. Cells growing in alginate were observed under the inverted microscope. Pathologic examination by hematoxylin and eosin staining was done at the end of the experiment. RESULTS: Migration of rapidly proliferating cells from the 3D scaffold and an inappropriate growth pattern were observed during the experiment. Cells and scaffold did not form a solid graft. CONCLUSIONS: The results obtained in this study indicated that alginate is not a good biomaterial for a durable implant.

Bone marrow progenitors from animals with chronic renal failure lack capacity of in vitro proliferation.

BACKGROUND: An interesting way to regenerate a kidney is an autologous bone marrow transplantation. The aim of this study was to examine whether chronic kidney disease influenced bone marrow progenitors. METHODS: Wistar male rats included group I (n = 4, chronic kidney disease 1/2, CKD 1/2) that underwent right nephrectomy. In group II (n = 3, chronic kidney disease 5/6, CKD 5/6) underwent removal of the right kidney and approximately one-third of the cortex of the left kidney. Animals in the control group (n = 4) were intact. Bone marrow cells obtained from femurs were separated using a CD34 Micro-Beads magnetic isolation kit. Isolated cells were counted using a trypan blue exclusion test. Numbers of isolated cells were presented as mean values with standard deviation with P < .05 considered significant. CD34(-) cells were cultivated and observed to the passage 6. RESULTS: The CKD rat model was used for in vitro experiments. There were no differences in cell numbers isolated from control rats versus both CKD rats. No differences were observed in CD34(-) cells after separation when compared to controls. Cell morphology was similar in primary CD34(-) cultures during the first days of primary culture. CD34(-) primary cultures established from chronic renal failure rats collapsed within 2 weeks. No differences were found in CD34(+) cell number after isolation when compared with controls. These cells did not form a monolayer. Cells in cultures established from control animals resembled normal fibroblast-like morphology of mesenchymal stem cells during 3 months. CONCLUSIONS: Bone marrow cells from chronic renal failure rats showed no capacity for in vitro proliferation. We speculated that bone marrow cells obtained from renal chronic failure patients may not be useful for autologous cell transplantation.

Progenitor cells are responsible for formation of human prostate epithelium primary cultures.

AIM: To analyze cell viability and morphology of primary cell cultures from CD133 immunolabeled and sorted cells from epithelium of patients suffering from benign prostate hyperplasia (BPH). METHODS: Cells obtained from 5 patients were divided in two fractions. First fraction (CD133+/CD133-) was cultivated in DMEM with 10% FBS. Second fraction was mixed with CD133 microbeads and immunomagnetically divided into CD133+ and CD133- fractions. These cells were cultivated and followed-up for 2 weeks. Cells were stained for Annexin V FITC/propidium iodide. RESULTS: Seventy CD133+/CD133- cultures, thirty-one of CD133+ and thirty-one of CD133- cells were established. There were 5-fold and 3-fold increase of CD133+/CD133- and CD133+ cell number after 2 weeks, respectively. CD133+/CD133- and CD133+ monolayers displayed epithelial-like morphology and cytokeratine expression. CD133- cultures collapsed. Cell viability within CD133+ and CD133- populations was 90.1-/+6.3% and 24.3-/+6.2%, respectively. Apoptotic index was 9.0-/+6.1% and 28.5-/+23.8% within CD133+ and CD133- cultures, respectively. CONCLUSIONS: CD133 separated human primary epithelial cell cultures displayed differences in morphology, viability and apoptosis occurrence. Immunomagnetic sorting can be recommended in each in vitro experiments with primary cell cultures in order to provide more objective results.

The influence of alpha1-antagonist on the expression pattern of TNF receptor family in primary culture of prostate epithelial cells from BPH patients.

Doxazosin triggers apoptosis via an imprecisely defined receptor mechanism that is related to tumor necrosis factor receptors (TNFRs). The aim of this study was to determine CD95, TNFR-1, TNFR-2, CD40 expression in primary prostate epithelial cultures incubated with doxazosin. Epithelial cultures were cultivated from 10 benign prostate hyperplasia patients. The cells were incubated with 20, 50 and 80 microM of doxazosin. Apoptosis was confirmed by fluorescence microscopy and flow cytometry. The cells were analyzed for expression of FAS, CD40, TNFR-1 and TNFR-2 by flow cytometry. Early apoptotic cells were present in all groups. A positive correlation was noticed between doxazosin dose and TNFR-1-, -2-positive cells. A decrease of CD40-positive cell population was observed in the lowest concentration. A decrease of mean fluorescence intensity signal of CD40 and CD95 was observed in the lowest concentration. Doxazosin-triggered apoptosis was dose-independent. The initiation of apoptosis was a result of receptors 'crosstalk' rather than a single receptor pathway activation. TNF receptor self-assembly process should be checked as a potential mechanism leading to apoptosis after doxazosin treatment.