Expression of BCL2, TP53, FOXA1, and GATA3 in pTa bladder cancer recurrence.

OBJECTIVES: In this study, we analyzed pTa bladder cancer (BC) for molecular markers BCL2, TP53, FOXA1, and GATA3 in relation to cancer recurrence. METHODS: We analyzed samples of 79 patients with the pTa stage of BC using a real-time polymerase chain reaction (real-time PCR) between September 2018 and September 2020. The expression levels of BCL2, TP53, FOXA1, and GATA3 were compared with homologous non-tumor bladder tissue. RESULTS: Expression of FOXA1, GATA3, and TP53 was significantly higher (p<0.01) in NMIBC samples compared to homologous non-tumor tissue. The expression of TP53 and FOXA1 in pTa was significantly lower (p<0.01) in the high-grade (HG) tumor when compared to the low-grade (LG) tumor. In contrast, the relative quantification (RQ) of GATA3 was significantly higher (p<0.01) in HG pTa. Patients with recurrence (pTa=33) had significantly higher expression of TP53, and GATA3 (p<0.01), and the gene of FOXA1 (p<0.01) had a significantly lower expression when compared to pTa tumors without recurrence. The expression of Bcl-2 was not statistically significant. CONCLUSION: Our results, indicate, that comparing expression levels of these genes in cancer and cancer-free tissue could provide valuable data, as patients with pTa BC recurrence within up to 54 months of follow-up had a significantly higher RQ of TP53, GATA3, and FOXA1 when compared to pTa BC patients without recurrence (Tab. 2, Fig. 8, Ref. 54). Text in PDF www.elis.sk Keywords: bladder cancer, gene expression, recurrence, GATA3, FOXA1, TP53, BCL2.

The diagnostic significance of C4d deposits, as an immunohistochemical proof of complement activation, in kidney glomerular pathologies and kidney transplantation.

C4d, a split product of C4 activation in classical and lectin pathways of the complement system activation, has been regarded as a footprint of tissue damage in antibody-mediated rejection in transplantology. The introduction of C4d staining into daily clinical practice aroused an ever-increasing interest in the role of antibody-mediated mechanisms in kidney allograft rejection. However, this marker of complement activation is also important in other various kidney glomerular pathologies such as immunoglobulin A nephropathy, membranoproliferative glomerulonephritis, lupus nephritis, and others. In routine histopathological practice, C4d staining can be done by two histological methods, specifically by immunofluorescence on frozen tissue using monoclonal antibody to C4d (with the downside of unsteady availability of frozen tissue) or by immunohistochemistry using C4d antibodies on formalin-fixed and paraffin-embedded renal tissue. The aim of this narrative review is to summarize recent knowledge about the complement fragment C4d and its significance in different kidney pathologies, focusing on its immunohistochemical detection in renal tissue biopsies. We have supplemented this review with our experience with our proprietary methodology of preparation and practical use of antibodies such as anti-C4d, on a small national level. Immunohistochemical staining for C4d has revolutionized the field of renal histopathology. Despite being a simple diagnostic test, its utility can be of utmost importance, especially in a resource-poor setting where immunofluorescence and frozen tissue may not be available (Fig. 2, Ref. 53). Keywords: C4d deposition, immunohistochemistry, kidney glomerular diseases, kidney transplant, renal tubular damage.

Current look at the most promising proteomic and glycomic biomarkers of bladder cancer.

BACKGROUND: Bladder cancer (BC) belongs to the most frequent cancer types. The diagnostic process is still long and costly, with a high percentage of false-positive or -negative results. Due to the cost and lack of effectiveness, older methods need to be supplemented or replaced by a newer more reliable method. In this regard, proteins and glycoproteins pose high potential. METHODS: We performed an online search in PubMed/Medline, Scopus, and Web of Science databases to find relevant studies published in English up until May 2023. If applicable, we set the AUC threshold to 0.90 and sensitivity/specificity (SN/SP) to 90%. FINDINGS: Protein and glycoprotein biomarkers are a demonstrably viable option in BC diagnostics. Cholinesterase shows promise in progression-free survival. BLCA-4, ORM-1 along with HTRA1 in the detection of BC. Matrix metallopeptidase 9 exhibits potential for stratification of muscle-invasive subtypes with high negative predictive value for aggressive phenotypes. Distinguishing non-muscle invasive subtypes benefits from Keratin 17. Neu5Gc-modified UMOD glycoproteins pose potential in BC diagnosis, while fibronectin, laminin-5, collagen type IV, and lamprey immunity protein in early detection of BC.

Bioadhesive and Injectable Hydrogels and Their Correlation with Mesenchymal Stem Cells Differentiation for Cartilage Repair: A Mini-Review.

Injectable bioadhesive hydrogels, known for their capacity to carry substances and adaptability in processing, offer great potential across various biomedical applications. They are especially promising in minimally invasive stem cell-based therapies for treating cartilage damage. This approach harnesses readily available mesenchymal stem cells (MSCs) to differentiate into chondrocytes for cartilage regeneration. In this review, we investigate the relationship between bioadhesion and MSC differentiation. We summarize the fundamental principles of bioadhesion and discuss recent trends in bioadhesive hydrogels. Furthermore, we highlight their specific applications in conjunction with stem cells, particularly in the context of cartilage repair. The review also encompasses a discussion on testing methods for bioadhesive hydrogels and direct techniques for differentiating MSCs into hyaline cartilage chondrocytes. These approaches are explored within both clinical and laboratory settings, including the use of genetic tools. While this review offers valuable insights into the interconnected aspects of these topics, it underscores the need for further research to fully grasp the complexities of their relationship.

Tissue Engineering and Stem Cell Therapy in Neurogenic Bladder Dysfunction: Current and Future Perspectives.

Tissue engineering (TE) is a rapidly evolving biomedical discipline that can play an important role in treating neurogenic bladder dysfunction and compensating for current conventional options' shortcomings. This review aims to analyze the current status of preclinical and clinical trials and discuss what could be expected in the future based on the current state of the art. Although most preclinical studies provide promising results on the effectiveness of TE and stem cell therapies, the main limitations are mainly the very slow translation of preclinical trials to clinical trials, lack of quality research on neurogenic preconditions of neurogenic bladder dysfunction outside of the spinal cord injury and varying therapeutic methods of the existing research that lacks a standardized approach.

Molecular classification of urothelial bladder carcinoma.

Urothelial bladder carcinoma (UC) ranks among the top ten most commonly diagnosed cancers worldwide on an annual basis. The standardized classification system for urothelial bladder tumors is the Tumor, Node, Metastasis classification, which reflects differences between non-muscle-invasive bladder carcinoma (NMIBC) and muscle-invasive bladder carcinoma (MIBC) and it depends on the extent to which tumor has infiltrated the bladder wall and other tissues and organs. NMIBC and MIBC exhibit great intrinsic heterogeneity regarding different prognoses, survival, progression, and treatment outcomes. In recent years, studies based on mRNA expression profiling revealed the existence of biologically relevant molecular subtypes of UC, which show variant molecular features that can provide more precise stratification of UC patients. Here, we present a complex classification of UC based on mRNA expression studies and molecular subtypes of NMIBC and MIBC in detail with regard to different mRNA expression profiles, mutational signatures, and infiltration by non-tumor cells. The possible impact of molecular subtyping on treatment decisions and patients' outcomes is outlined, too.

Resorbable Biomaterials Used for 3D Scaffolds in Tissue Engineering: A Review.

This article provides a thorough overview of the available resorbable biomaterials appropriate for producing replacements for damaged tissues. In addition, their various properties and application possibilities are discussed as well. Biomaterials are fundamental components in tissue engineering (TE) of scaffolds and play a critical role. They need to exhibit biocompatibility, bioactivity, biodegradability, and non-toxicity, to ensure their ability to function effectively with an appropriate host response. With ongoing research and advancements in biomaterials for medical implants, the objective of this review is to explore recently developed implantable scaffold materials for various tissues. The categorization of biomaterials in this paper includes fossil-based materials (e.g., PCL, PVA, PU, PEG, and PPF), natural or bio-based materials (e.g., HA, PLA, PHB, PHBV, chitosan, fibrin, collagen, starch, and hydrogels), and hybrid biomaterials (e.g., PCL/PLA, PCL/PEG, PLA/PEG, PLA/PHB PCL/collagen, PCL/chitosan, PCL/starch, and PLA/bioceramics). The application of these biomaterials in both hard and soft TE is considered, with a particular focus on their physicochemical, mechanical, and biological properties. Furthermore, the interactions between scaffolds and the host immune system in the context of scaffold-driven tissue regeneration are discussed. Additionally, the article briefly mentions the concept of in situ TE, which leverages the self-renewal capacities of affected tissues and highlights the crucial role played by biopolymer-based scaffolds in this strategy.

Decellularization of the human urethra for tissue engineering applications.

Recently, several scaffolds have been introduced for urethral tissue engineering. However, acellular human urethral scaffold harvested from deceased donors may provide significant advantages compared to synthetic, composite, or other biological scaffolds. This study aims to develop the protocol for decellularization of the human urethra that preserves substantial extracellular matrix (ECM) components, which are essential for subsequent recellularization mimicking the natural environment of the native ECM. A total of 12 human urethras were harvested from deceased donors. An equal part of every harvested urethra was used as a control sample for analyses. The protocol design was based on the enzyme-detergent-enzyme method. Trypsin and Triton X-100 were used to remove cells, followed by DNase treatment to remove DNA residues. Subsequently, the specimens were continually rinsed in deionized water for seven days. The efficiency of decellularization was determined by histochemistry, immunohistochemical staining, scanning electron microscopy (SEM), and DNA quantification. Histological analysis confirmed cell removal and preservation of urethral structure after decellularization. The preservation of collagen IV and fibronectin was confirmed by histologic examination and immunohistochemical staining. SEM confirmed the maintenance of the ultrastructural architecture of ECM and fibers. DNA content in decellularized urethra was significantly lower compared to the native sample (P < 0.001), and so the criteria for decellularized tissue were met. Cytotoxicity analysis data showed that the matrix-conditioned medium did not contain soluble toxins and had no significant inhibitory effect on cell proliferation, providing evidence that the decellularized samples are not toxic. This study demonstrates the feasibility of the enzyme-detergent-enzyme-based decellularization protocol for removing cellular components and maintaining urethral ECM and its ultrastructure. Moreover, obtained results provide solid ground for recellularization and urethral tissue engineering, which will follow.

The Role of Extracellular Matrix and Hydrogels in Mesenchymal Stem Cell Chondrogenesis and Cartilage Regeneration.

Diseases associated with articular cartilage disintegration or loss are still therapeutically challenging. The traditional treatment approaches only alleviate the symptoms while potentially causing serious side effects. The limited self-renewal potential of articular cartilage provides opportunities for advanced therapies involving mesenchymal stem cells (MSCs) that are characterized by a remarkable regenerative capacity. The chondrogenic potential of MSCs is known to be regulated by the local environment, including soluble factors and the less discussed extracellular matrix (ECM) components. This review summarizes the process of chondrogenesis, and also the biological properties of the ECM mediated by mechanotransduction as well as canonical and non-canonical signaling. Our focus is also on the influence of the ECM's physical parameters, molecular composition, and chondrogenic factor affinity on the adhesion, survival, and chondrogenic differentiation of MSCs. These basic biological insights are crucial for a more precise fabrication of ECM-mimicking hydrogels to improve cartilage tissue reconstruction. Lastly, we provide an overview of hydrogel classification and characterization. We also include the results from preclinical models combining MSCs with hydrogels for the treatment of cartilage defects, to support clinical application of this construct. Overall, it is believed that the proper combination of MSCs, hydrogels, and chondrogenic factors can lead to complex cartilage regeneration.

Biomarkers of Bladder Cancer: Cell-Free DNA, Epigenetic Modifications and Non-Coding RNAs.

Bladder cancer (BC) is the 10th most frequent cancer in the world. The initial diagnosis and surveillance of BC require a combination of invasive and non-invasive methods, which are costly and suffer from several limitations. Cystoscopy with urine cytology and histological examination presents the standard diagnostic approach. Various biomarkers (e.g., proteins, genes, and RNAs) have been extensively studied in relation to BC. However, the new trend of liquid biopsy slowly proves to be almost equally effective. Cell-free DNA, non-coding RNA, and other subcellular structures are now being tested for the best predictive and diagnostic value. In this review, we focused on published gene mutations, especially in DNA fragments, but also epigenetic modifications, and non-coding RNA (ncRNA) molecules acquired by liquid biopsy. We performed an online search in PubMed/Medline, Scopus, and Web of Science databases using the terms "bladder cancer", in combination with "markers" or "biomarkers" published until August 2022. If applicable, we set the sensitivity and specificity threshold to 80%. In the era of precision medicine, the development of complex laboratory techniques fuels the search and development of more sensitive and specific biomarkers for diagnosis, follow-up, and screening of BC. Future efforts will be focused on the validation of their sensitivity, specificity, predictive value, and their utility in everyday clinical practice.

Tissue Engineering of the Urethra: From Bench to Bedside.

Tissue engineering (TE) is a promising approach for repair/substitution of damaged tissues and organs. Urethral strictures are common and serious health conditions that impair quality of life and may lead to serious organ damage. The search for ideal materials for urethral repair has led to interest of scientists and surgeons in urethral TE. Over the last decades, a significant amount of preclinical studies and considerable progress have been observed. In contrast, urethral TE has made slow progress in clinical practice so far. To address this, we conducted a systematic review of the literature on clinical applications of TE constructs for urethral repair in the last three decades. In summary, the TE approach is promising and effective, but many issues remain that need to be addressed for broader adoption of TE in urethral repair. Better design of trials, better cooperation of research groups and centralization could lead to reduction of costs and slowly proceed to commercialization and routine use of TE products for urethral reconstruction.

Isolation, Culture and Comprehensive Characterization of Biological Properties of Human Urine-Derived Stem Cells.

Mesenchymal stem cells (MSCs) represent an attractive source within the field of tissue engineering. However, their harvesting often requires invasive medical procedures. Urine-derived stem cells (UDSCs) display similar properties to MSCs, and their obtention and further processing is non-invasive for the donors as well as low cost. Here, we offer a comprehensive analysis of their biological properties. The goal of this study was to analyze their morphology, stemness, differentiation potential and cytokine profile. We have successfully isolated UDSCs from 25 urine samples. First colonies emerged up to 9 days after the initial seeding. Cell doubling time was 45 ± 0.24 SD, and when seeded at the density of 100 cells/cm2, they formed 42 ± 6.5 SD colonies within 10 days. Morphological analyzes revealed that two different types of the cell populations have been present. The first type had a rice-grain shape and the second one was characterized by a polyhedral shape. In several cell cultures, dome-shaped cells were observed as well. All examined UDSCs expressed typical MSC-like surface markers, CD73, CD90 and CD105. Moreover, conditioned media from UDSCs were harvested, and cytokine profile has been evaluated showing a significantly higher secretory rate of IL-8, IL-6 and chemokines MCP-1 and GM-CSF. We have also successfully induced human UDSCs into chondrogenic, osteogenic and myogenic cell lineages. Our findings indicate that UDSCs might have immense potential in the regeneration of the damaged tissues.

Cardiac Telocytes 16 Years on-What Have We Learned So Far, and How Close Are We to Routine Application of the Knowledge in Cardiovascular Regenerative Medicine?

The regeneration of a diseased heart is one of the principal challenges of modern cardiovascular medicine. There has been ongoing research on stem-cell-based therapeutic approaches. A cell population called telocytes (TCs) described only 16 years ago largely contributed to the research area of cardiovascular regeneration. TCs are cells with small bodies and extremely long cytoplasmic projections called telopodes, described in all layers of the heart wall. Their functions include cell-to-cell signaling, stem-cell nursing, mechanical support, and immunoregulation, to name but a few. The functional derangement or quantitative loss of TCs has been implicated in the pathogenesis of myocardial infarction, heart failure, arrhythmias, and many other conditions. The exact pathomechanisms are still unknown, but the loss of regulative, integrative, and nursing functions of TCs may provide important clues. Therefore, a viable avenue in the future modern management of these conditions is TC-based cell therapy. TCs have been previously transplanted into a mouse model of myocardial infarction with promising results. Tandem transplantation with stem cells may provide additional benefit; however, many underresearched areas need to be addressed in future research before routine application of TC-based cell therapy in human subjects. These include the standardization of protocols for isolation, cultivation, and transplantation, quantitative optimization of TC transplants, cost-effectivity analysis, and many others.

In Vitro Characterization of Poly(Lactic Acid)/ Poly(Hydroxybutyrate)/ Thermoplastic Starch Blends for Tissue Engineering Application.

Complex in vitro characterization of a blended material based on Poly(Lactic Acid), Poly(Hydroxybutyrate), and Thermoplastic Starch (PLA/PHB/TPS) was performed in order to evaluate its potential for application in the field of tissue engineering. We focused on the biological behavior of the material as well as its mechanical and morphological properties. We also focused on the potential of the blend to be processed by the 3D printer which would allow the fabrication of the custom-made scaffold. Several blends recipes were prepared and characterized. This material was then studied in the context of scaffold fabrication. Scaffold porosity, wettability, and cell-scaffold interaction were evaluated as well. MTT test and the direct contact cytotoxicity test were applied in order to evaluate the toxic potential of the blended material. Biocompatibility studies were performed on the human chondrocytes. According to our results, we assume that material had no toxic effect on the cell culture and therefore could be considered as biocompatible. Moreover, PLA/PHB/TPS blend is applicable for 3D printing. Printed scaffolds had highly porous morphology and were able to absorb water as well. In addition, cells could adhere and proliferate on the scaffold surface. We conclude that this blend has potential for scaffold engineering.

Expression of E-cadherin, Ki-67, and p53 in urinary bladder cancer in relation to progression, survival, and recurrence.

Although the incidence varies with age and gender, urothelial bladder cancer is a relatively frequently occurring malignancy with variable clinical behavior that often has high recurrence rates. In this study, we analyzed the tumor tissues of 224 patients with pTa, pT1, and pT2 urinary bladder cancer. We performed a histomorphologic analysis and immunohistochemistry for p53, Ki-67, and E-cadherin, which were selected as markers of the malignant process. For pTa and pT1, univariate analyses of cancer-specific survival (CSS), progression-free survival (PFS), and recurrence-free survival (RFS) were calculated using the Kaplan-Meier method, the log-rank test and Cox regression. Multivariate analysis was performed by a Cox regression analysis. Ki-67 (P<0.001) was significantly associated with CSS, but the highest association was shown for E-cadherin (P<0.001). For pT1 and pTa, the Kaplan-Meier analysis and the log-rank test revealed significantly worse PFS for patients with higher levels of Ki-67 (P<0.001) and lower levels of E-cadherin (P<0.001). Based on these obtained results, it can be clearly stated that Ki-67 and E-cadherin expression levels are associated with CSS, PFS and RFS. The clinical utility of these markers is valuable for pTa and pT1 urinary bladder cancer and should be further verified with prospective multi-center trials.

Bioengineered Scaffolds as Substitutes for Grafts for Urethra Reconstruction.

Urethral defects originating from congenital malformations, trauma, inflammation or carcinoma still pose a great challenge to modern urology. Recent therapies have failed many times and have not provided the expected results. This negatively affects patients' quality of life. By combining cells, bioactive molecules, and biomaterials, tissue engineering can provide promising treatment options. This review focused on scaffold systems for urethra reconstruction. We also discussed different technologies, such as electrospinning and 3D bioprinting which provide great possibility for the preparation of a hollow structure with well-defined architecture.

Cells Involved in Urethral Tissue Engineering: Systematic Review.

The urethra is part of the lower urinary tract and its main role is urine voiding. Its complex histological structure makes urethral tissue prone to various injuries with complicated healing processes that often lead to scar formation. Urethral stricture disease can affect both men and women. The occurrence of this pathology is more common in men and thus are previous research has been mainly oriented on male urethra reconstruction. However, commonly used surgical techniques show unsatisfactory results because of complications. The new and progressively developing field of tissue engineering offers promising solutions, which could be applied in the urethral regeneration of both men´s and women´s urethras. The presented systematic review article offers an overview of the cells that have been used in urethral tissue engineering so far. Urine-derived stem cells show a great perspective in respect to urethral tissue engineering. They can be easily harvested and are a promising autologous cell source for the needs of tissue engineering techniques. The presented review also shows the importance of mechanical stimuli application on maturating tissue. Sufficient vascularization and elimination of stricture formation present the biggest challenges not only in customary surgical management but also in tissue-engineering approaches.

Tissue engineering of urethra: Systematic review of recent literature.

The purpose of this article was to perform a systematic review of the recent literature on urethral tissue engineering. A total of 31 articles describing the use of tissue engineering for urethra reconstruction were included. The obtained results were discussed in three groups: cells, scaffolds, and clinical results of urethral reconstructions using these components. Stem cells of different origin were used in many experimental studies, but only autologous urothelial cells, fibroblasts, and keratinocytes were applied in clinical trials. Natural and synthetic scaffolds were studied in the context of urethral tissue engineering. The main advantage of synthetic ones is the fact that they can be obtained in unlimited amount and modified by different techniques, but scaffolds of natural origin normally contain chemical groups and bioactive proteins which increase the cell attachment and may promote the cell proliferation and differentiation. The most promising are smart scaffolds delivering different bioactive molecules or those that can be tubularized. In two clinical trials, only onlay-fashioned transplants were used for urethral reconstruction. However, the very promising results were obtained from animal studies where tubularized scaffolds, both non-seeded and cell-seeded, were applied. Impact statement The main goal of this article was to perform a systematic review of the recent literature on urethral tissue engineering. It summarizes the most recent information about cells, seeded or non-seeded scaffolds and clinical application with respect to regeneration of urethra.

The assessment and evaluation of low-frequency noise near the region of infrasound.

The main aim of this paper is to present recent knowledge about the assessment and evaluation of low-frequency sounds (noise) and infrasound, close to the threshold of hearing, and identify their potential effect on human health and annoyance. Low-frequency noise generated by air flowing over a moving car with an open window was chosen as a typical scenario which can be subjectively assessed by people traveling by automobile. The principle of noise generated within the interior of the car and its effects on the comfort of the driver and passengers are analyzed at different velocities. An open window of a car at high velocity behaves as a source of specifically strong tonal low-frequency noise which is generally perceived as annoying. The interior noise generated by an open window of a passenger car was measured under different conditions: Driving on a highway and driving on a typical roadway. First, an octave-band analysis was used to assess the noise level and its impact on the driver's comfort. Second, a fast Fourier transform (FFT) analysis and one-third octave-band analysis were used for the detection of tonal low-frequency noise. Comparison between two different car makers was also done. Finally, the paper suggests some possibilities for scientifically assessing and evaluating low-frequency sounds in general, and some recommendations are introduced for scientific discussion, since sounds with strong low-frequency content (but not only strong) engender greater annoyance than is predicted by an A-weighted sound pressure level.

Varicocele treatment: a 2-centers comparison between non microsurgical open correction, laparoscopic approach and retrograde percutaneous sclerotization on 463 cases.

OBJECTIVES: To determine whether there are differences in sperm parameters improvement after different varicocele correction techniques. To determine the role of age in sperm parameters improvement. METHODS: 2 different European centers collected pre- and postoperative sperm parameters of patients undergoing varicocele correction. Among 463 evaluated patients, 367 were included. Patients were divided in procedure-related and age-related groups. Ivanissevich inguinal open surgical procedure (OS), lymphatic-sparing laparoscopic approach (LSL) and retrograde percutaneous transfemoral sclerotization (RPS) were performed. As outcome measurements sperm count (millions/mL, SC) and percentage of mobile sperms were analyzed. Univariate and multivariate regression between the defined groups; bivariate regression analysis between age and sperm count and motility. RESULTS: Number of patients: OS 78; LSL 85; RPS 204. Mean age 30.2 (SD 6.83); postoperative SC increased from 18.2 to 30.1 (CI 95% 27.3-32.9; p < 0,001); motility from 25.6 to 32.56% (30.9-34.2; p < 0.001). OS: SC varied from 16.9 to 18.2 (p < 0.001); sperm motility from 29% to 33% (p < 0.001). LSL: SC from 15.5 to 17.2 (p < 0.001); motility from 27 to 31% (p < 0.001). RPS: SC from 18.9 to 36.2 (p < 0.001); motility from 24% to 32% (p < 0.001). Univariate and multivariate analysis confirmed the significant difference of SC variation in RPS, compared to the other groups (p < 0.001). No significance between LSL and OS (p = 0.826). No significant differences regarding motility (p = 0.8). CONCLUSIONS: Varicocele correction is confirmed useful in improving sperm parameters; sclerotization technique leads to a better sperm improvement compared to other studied procedures; improvement in seminal parameters is not affected by age of the patients treated.