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.

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.

Innervation density and types of nerves in prostate cancer.

Innervation of cancerous tissue represents an important pathway enabling the nervous system to influence the processes associated with the initiation, progression, and metastasis of a neoplastic process. In the context of prostate cancer, several papers report the presence of innervation and its modulating effect on the cancer prognosis. However, most of the data are experimental, with limited information on human prostate cancer innervation. Morphometric analysis of archival prostate specimen immunohistochemistry with neural markers PGP9.5 and S100 showed a significant decrease of nerve density in the prostate cancer (n=44) compared to the normal prostate tissue (n=18) and benign prostatic hyperplasia (n=28). Sympathetic nerves were detected with TH, parasympathetic with VAChT, and sensory nerves with SP and CGRP protein detection. Dual immunofluorescence revealed numerous sympathetic nerves in normal prostate and benign prostatic hyperplasia, especially in the peripheral parts. Only a few parasympathetic nerves were found between the glands and in the peripheral parts of the prostate and benign hyperplasia. Sporadic positivity for sensory innervation was present only in approximately 1/10 of nerve fibers, especially in the larger nerves. The pattern of innervation in prostate cancer was analogous to that in normal prostate gland and benign prostatic hyperplasia but there was a significantly lower amount of all nerve types, especially in high-grade carcinoma cases. Although not significant, there was a tendency of decreasing innervation density with increasing Gleason score. Regarding the low density of nerves in prostate carcinoma, the significantly lower PCNA counts in nerves of the cancer specimens cannot be ascribed to lower proliferation activity. Our data confirmed the lower nerve density in the prostate cancer compared to the benign prostate tissue. We could not approve an increased nerve proliferation activity in prostate cancer. All nerve types, most the sympathetic, less the parasympathetic, and the sensory nerves, are present in prostate cancer. The highest nerve density at the periphery of the cancer tissue implies this to be the result of an expansive tumor growth. It is evident that the results of experimental prostate cancer models can be applied to human pathology only to a certain extent. The relation between the range of innervation and the biology of prostate cancer is very complex and will require more detailed information to be applied in therapeutic solutions.

Improvement in cardiovascular risk markers by the combined effect of natural polyphenols and vitamins in patients after kidney transplantation.

OBJECTIVES: The aim of this study was to evaluate the potential of supportive therapy by natural polyphenols combined with vitamins C and E on kidney function and risk factors of cardiovascular diseases in renal transplant recipients (RTR). BACKGROUND: Transplant patients have an altered lipid profile associated with the development of cardiovascular disease, which is a major cause of graft loss and mortality in patients. METHODS: The study included 29 renal transplant recipients with mean graft function levels. The lipoprotein (atherogenic and non-atherogenic) subfractions were identified and quantified in plasma by polyacrylamide gel electrophoresis. RESULTS: After supplementation, glomerular filtration rate (GFR) was increased by 8 %, serum creatinine was decreased by 6.7 % and significant changes were found in atherogenic LDL subfractions. The effect of supplementation was observed in arylesterase and lactonase activities of paraoxonase 1 which increased by 9.3 % and 8.1 %, respectively. In addition, significantly decreased levels of neopterin (by 16 %) and asymmetric dimethylarginine (ADMA) (by 7.9 %) were found. CONCLUSION: We could summarize that supportive therapy improves the renal function (GFR, serum creatinine), and reduces the risk of cardiovascular disease by affecting important risk markers of atherosclerosis (lipid profile, paraoxonase 1 activity, neopterin and ADMA) in RTR (Tab. 4, Fig. 1, Ref. 53).

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.

Expression of HLA-G transcripts in graft biopsy samples of renal transplant recipients.

BACKGROUND: The HLA-G molecule has a high potential to modulate immune response towards the improvement of graft survival after transplantation. In this work, we have analyzed the total HLA-G mRNA expression in graft tissues of dysfunctional transplanted kidneys. MATERIAL AND METHODS: We examined 84 kidney biopsy samples obtained from 65 renal transplant recipients with dysfunctional graft (50 males, 15 females; average age 46.8 ± 11.9 years). 52 specimens were with signs of acute rejection and 32 without any rejection characteristics (diagnosed as glomerulonephritis, ATN and IFTA). Patients with acute rejection were divided into three groups: antibody-mediated rejection (AMR; n = 23), T cell mediated rejection (TCMR; n = 16) and combined antibody and T cell-mediated rejection (AMR + TCMR; n=13). The biopsy samples were taken from a dysfunctional graft at different time periods after kidney transplantation. The relative expression of total HLA-G mRNA in biopsy specimens was determined by real time RT-PCR. The correlation between HLA-G mRNA expression and dysfunctional graft state was investigated. The impact of different factors (post-transplantation interval, gender,mismatch, induction therapy and cold ischemia time) on relative expression of total HLA-G mRNA was also studied. RESULTS: We have found that the levels of HLA-G transcripts in kidneys with rejection were higher than those in non-rejected but dysfunctional grafts (P = 0.0003). The highest levels of HLA-G mRNA were detected at combined AMR + TCMR rejection (P= 0.005). The time-course analysis of total HLA-G mRNA expression was also studied. In both dysfunctional graft groups (rejected and non-rejected) the lower levels of HLA-G transcripts were detected during early post-transplant period (1­3 months), however a substantial increase of HLA-G mRNA expression was observed after an extended period of time(N3 months). It was also revealed that antibody induction therapy may reduce HLA-G expression (P=0.0004) and in female samples were higher levels of HLAG transcripts than those in male recipients (P=0.003). It was found no significant impact of age, cold ischemic time, PRA (Panel Reactive Antibody) score, and a number of HLA-mismatches on HLA-G mRNA expression. CONCLUSIONS: We have demonstrated that the expression of total HLA-GmRNA in renal grafts can be influenced by different factors such as clinical state of transplanted kidney, elapsed time after transplantation, gender and antibody induction therapy. We have proved that HLA-G mRNA expression was significantly higher in recipients with acute rejection in comparison to patients with dysfunctional but non-rejected grafts.

The effect of natural polyphenols on the oxidative stress markers in patients with diabetic nephropathy.

BACKGROUND: Nephropathy remains a significant cause of morbidity and mortality in the diabetic population and is the leading cause of end-stage renal failure, hence kidney transplantations. As a result of the diabetic milieu, increased generation of reactive oxygen species is thought to play a key role in the progression of diabetic nephropathy. Aim of this study was to evaluate the effect of Pycnogenol (Pyc), the extract from Pinus pinaster, on the level of glucose, advanced glycation end products (AGE) and oxidative stress markers in patients with Diabetic nephropathy (DN). SUBJECT AND METHODS: To double blind randomised placebo controlled exploratory study were enrolled 20 men with DN received daily Pyc (120mg) or a placebo. Patients were investigated before, one and three months after Pyc administration and after termination of drugs supplementation. RESULTS: The level of glucose, AGEs, malondialdehyde (MDA), 8-isoprostanes (8-Iso), as well as protein carbonyls (PC) were increased in comparison to control group. We have found lower level of glucose, AGEs, MDA and 8-Iso after 3 months of Pyc administration in comparison to the beginning state and to placebo group, but these results did not reach significance. CP and TAS were not affected. CONCLUSION: Our data allow us to conclude that Pycnogenoladministration reduces oxidative damage to lipids rather through the decreased glucose level than through the influence of antioxidant capacity of plasma. This study was supported by grant Ministry of Health 2012/8-ukba-8.