Publications by authors named "Melanie von Brandenstein"

24 Publications

  • Page 1 of 1

Unraveling Subcellular and Ultrastructural Changes During Vitrification of Human Spermatozoa: Effect of a Mitochondria-Targeted Antioxidant and a Permeable Cryoprotectant.

Front Cell Dev Biol 2021 2;9:672862. Epub 2021 Jul 2.

Department of Obstetrics and Gynaecology, Medical Faculty, Cologne University, Cologne, Germany.

Mitochondria-targeted antioxidants have great potential to counterbalance the generated reactive oxygen species (ROS) because they cross the inner membrane of the mitochondria. Still, their use was not reported in vitrified human spermatozoa. Our laboratory has successfully vitrified spermatozoa without the use of permeable cryoprotectants, but subcellular-level evidence was missing. Therefore, this study aimed to improve spermatozoa vitrification using a mitochondria-targeted antioxidant (mitoquinone, MitoQ), reveal ultrastructural changes in the spermatozoa due to the use of a permeable cryoprotectant, and report alterations of functional proteins during the spermatozoa vitrification process. For this, each of 20 swim-up-prepared ejaculates was divided into seven aliquots and diluted with a vitrification medium supplemented with varying concentrations of MitoQ (0.02 and 0.2 μM), glycerol (1, 4, and 6%), and a combination of MitoQ and glycerol. All aliquots were vitrified by the aseptic capillary method developed in our laboratory. The spermatozoa function assays revealed that the addition of either MitoQ (0.02 μM), glycerol (1%), or a combination of MitoQ (0.02 μM) and glycerol (1%) in the vitrification medium results in better or equivalent spermatozoa quality relative to the control. Transmission electron microscopy revealed that MitoQ protects the spermatozoa from undergoing ultrastructural alterations, but glycerol induced ultrastructural alterations during the vitrification process. Next, we performed label-free quantitative proteomics and identified 1,759 proteins, of which 69, 60, 90, and 81 were altered in the basal medium, 0.02 μM MitoQ, 1% glycerol, and Mito-glycerol groups, respectively. Actin, tubulins, and outer dense fiber proteins were not affected during the vitrification process. Some of the identified ubiquitinating enzymes were affected during spermatozoa vitrification. Only a few proteins responsible for phosphorylation were altered during vitrification. Similarly, several proteins involved in spermatozoa-egg fusion and fertilization (IZUMO1 and Tektin) were not affected during the vitrification process. In conclusion, MitoQ attenuates the vitrification-induced ultrastructural changes and alterations in the key proteins involved in spermatozoa functions and fertilization.
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http://dx.doi.org/10.3389/fcell.2021.672862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8284099PMC
July 2021

Teratomatous Elements in Orchiectomy Specimens Are Associated with a Reduced Relapse-Free Survival in Metastasized Testicular Germ Cell Tumors.

Urol Int 2021 Jun 15:1-7. Epub 2021 Jun 15.

Department of Urology, Uro-Oncology, Robot Assisted and Reconstructive Urologic Surgery, University Hospital Cologne, Cologne, Germany.

Introduction: The impact of teratomatous elements in orchiectomy specimens of metastasized testicular germ cell tumors (TGCT) regarding oncological outcome is still unclear.

Methods: We performed a retrospective analysis including 146 patients with metastasized TGCT analysing patient characteristics.

Results: Twenty-six (18%) of all patients showed teratomatous elements in the orchiectomy specimens. TGCT with teratomatous elements showed a significantly higher frequency of clinical-stage 2C-3 disease (73 vs. 49%, p = 0.031), visceral metastases (58 vs. 32%, p = 0.015), and poor prognosis (p = 0.011) than TGCT without teratomatous elements. Teratoma-containing TGCT revealed a significantly higher rate of post-chemotherapy retroperitoneal lymph node dissection (PC-RPLND, 54 vs. 32%, p = 0.041), with teratomatous elements being more often present in the PC-RPLND specimens (43 vs. 11%, p = 0.020) than nonteratoma-containing primaries. In the Kaplan-Meier estimates, the presence of teratomatous elements in orchiectomy specimens was associated with a significantly reduced relapse-free survival (RFS) (p = 0.049) during a median follow-up of 36 months (10-115.5).

Conclusions: The presence of teratomatous elements in orchiectomy specimens is associated with an advanced tumor stage, worse treatment response as well as a reduced RFS in metastasized TGCT. Consequently, the presence of teratomatous elements might act as a reliable stratification tool for treatment decision in TGCT patients.
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http://dx.doi.org/10.1159/000515715DOI Listing
June 2021

The combination of microRNA-371a-3p and 375-5p can distinguish viable germ cell tumor and teratoma from necrosis in postchemotherapy retroperitoneal lymph node dissection specimens.

Transl Androl Urol 2021 Apr;10(4):1647-1655

Department of Urology, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany.

Background: To identify a combination of microRNAs (miRNA) to differentiate between viable tumor (V) or teratoma (T) and necrosis/fibrosis (N) in pcRPLND specimens of metastatic germ cell tumor (GCT) patients with residual masses ≥1 cm after chemotherapy. Biomarker guided therapy could reduce overtreatment with pcRPLND in patients with only N.

Methods: We selected 48 metastatic GCT patients who had undergone pcRPLND. V, pure T and N was shown in the resected tissue of 16 patients, respectively. Of these areas total RNA was isolated and miRNA expression was analyzed for miR-371a-3p, 375-3p, and 375-5p using qPCR. ROC analysis was performed for each miRNA and for all combinations in order to determine the discriminatory capacity of V and T . N.

Results: On comparing V . N miR-371a-3p achieved the highest fold change (FC) of 31.1 (P=0.023) while for T . N miR-375-5p performed best (FC 64.2; P<0.001). Likewise, the most accurate AUC for V was 0.75 using miR-371a-3p, for T 0.80 using miR-375-5p. Combining the best performing miRNAs for V and T resulted in an AUC of 0.94 with a sensitivity of 93.75, specificity of 93.75, PPV of 96.8 and NPV of 83.3.

Conclusions: By combining miR-371a-3p and miR-375-5p in pcRPLND tissue samples V and T could be distinguished from necrosis/fibrosis with great accuracy. This combination of miRNAs might serve as new biomarker in the future, in order to spare miRNA-negative patients from pcRPLND. However, further studies analyzing patient's serum are needed to confirm the clinical impact of these biomarkers.
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http://dx.doi.org/10.21037/tau-20-1349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100847PMC
April 2021

Non-invasive urine markers for the differentiation between RCCs and oncocytoma.

J Clin Lab Anal 2021 May 7;35(5):e23762. Epub 2021 May 7.

Department of Urology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany.

Background: Recently, our group showed that Vim3 is overexpressed in tissue samples of renal oncocytomas and Mxi-2 in clear cell renal carcinoma (ccRCC). The mechanism leading to the truncation of both proteins is known and involves with two miRs, both detectable in urine. Since the analysis of miRs is time-consuming, our aim was to identify the truncated proteins in urine instead. Furthermore, urine samples from small renal masses (SRMs) (n = 45, <4 cm) were analyzed to get a pre-surgical differentiation of the cancer subtypes.

Methods: Urines were accessed from the urological biobank (n = 350). Proteins were isolated from urine samples, and Western blots were performed. Each sample was analyzed with ELISA for the expression of Vim3 and Mxi-2. A lateral flow assay was established. For the detection of SRMs, the miRs were isolated and qRT-PCR was performed.

Results: A significant increase of Vim3 in urines from patients with oncocytoma (n = 20) was detectable with ELISA compared to all other subtypes of RCCs (chromophobe (n = 50), papillary (n = 40), ccRCC (n = 200), and controls (n = 40) (***p < 0.0001)). Mxi-2 was predominantly overexpressed in ccRCCs (***p < 0.0001). Lateral flow assay of Vim3 and Mxi-2 shows two bands in the case of oncocytoma and ccRCC indicating the specificity of this test. For SRMs, an overexpression of miR-15a/Mxi2 was detectable in urine samples from ccRCC and chromoRCC patients. In contrast to that, miR-498/Vim3 were predominantly overexpressed in oncocytoma patients.

Conclusion: Both proteins (Vim3 and Mxi-2) were detectable in patients' urines and can be used for the non-invasive differentiation of kidney cancers.
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http://dx.doi.org/10.1002/jcla.23762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8128285PMC
May 2021

Cryo-banking of human spermatozoa by aseptic cryoprotectants-free vitrification in liquid air: Positive effect of elevated warming temperature.

Cell Tissue Bank 2021 Feb 19. Epub 2021 Feb 19.

Department of Obstetrics and Gynaecology, Medical Faculty, Cologne University, 50931, Cologne, Germany.

Cryoprotectant-free vitrification is a common method for spermatozoa cryopreservation by direct plunging into liquid nitrogen. However, the commercial liquid nitrogen could be potentially contaminated by microorganisms. Warming temperature plays an essential role for quality of human spermatozoa after vitrification. This study aimed to evaluate comparatively a quality spermatozoa after vitrification in liquid nitrogen and clean liquid air as well as with two warming rates: at 42 °C and 45 °C. After performing of routine swim-up of normozoospermia samples, spermatozoa from the same ejaculate were divided into two groups: vitrified in liquid nitrogen (LN) and sterile liquid air (LA). Spermatozoa of LN group were warmed at 42 °C, and spermatozoa of LA groups were divided and warmed at 42 °C (LA42) and 45 °C (LA45). Then spermatozoa motility, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), reactive nitrogen species (RNS), and viability were assessed. It was no found significant differences in quality of spermatozoa from LN and LA groups in the motility, ROS, MMP, RNS rates after warming at 42 °C. A tendency to obtain better spermatozoa quality was found with using of warming by 42 °C in comparison with 45 °C. It was concluded that cryoprotectant-free vitrification by direct dropping of human spermatozoa into clean liquid air can be used as an alternative to cooling in liquid nitrogen. Warming of spermatozoa at 42 °C allows to preserve the spermatozoa physiological parameters.
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http://dx.doi.org/10.1007/s10561-021-09904-0DOI Listing
February 2021

[Using preorchiectomy tumor marker serum concentrations for International Germ Cell Consensus Classification (IGCCCG) risk group assignment results in significant numbers of up- and downstaging].

Urologe A 2021 Mar 11;60(3):337-343. Epub 2021 Jan 11.

Klinik für Urologie, Uro-Onkologie, spezielle urologische und roboter-assistierte Chirurgie, Universitätsklinikum Köln, Kerpener Str. 62, 50937, Köln, Deutschland.

Background: The prognostic classification system of the International Germ Cell Cancer Cooperative Group (IGCCCG) for testicular germ cell tumors is based on the histological subtype, location of the primary tumor, extent of metastatic spread and prechemotherapy tumor marker serum concentrations.

Objectives: In this study, we aim to identify whether the use of preorchiectomy instead of prechemotherapy tumor marker serum concentration has an impact on IGCCCG risk group assignment.

Materials And Methods: We performed a retrospective analysis including 135 patients with metastasized testicular germ cell tumors. Analysis of the clinical information with a focus on the tumor marker serum concentration preorchiectomy and prechemotherapy was performed, thus leading to the grouping of patients according to IGCCCG risk group assignment.

Results: Using preorchiectomy instead of prechemotherapy tumor markers led to an incorrect IGCCCG risk group classification in 8% (11/135) of all patients, and consequently to a non-guideline concordant treatment. Up-staging was observed in 8 of 11 patients, representing 6% (8/135) of the total patient cohort. Three of the 11 misclassified patients showed a down-staging and thus describe 2% (3/135) of the total patient cohort.

Conclusions: Using preorchiectomy tumor markers instead of prechemotherapy serum concentration might lead to an incorrect IGCCCG risk group assignment as well as non-guideline concordant treatment. Consequently, prechemotherapy tumor marker serum concentration should be applied for guideline concordant staging of patients.
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http://dx.doi.org/10.1007/s00120-020-01432-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979643PMC
March 2021

Aseptic capillary vitrification of human spermatozoa: Cryoprotectant-free vs. cryoprotectant-included technologies.

Cryobiology 2021 04 7;99:95-102. Epub 2021 Jan 7.

Department of Obstetrics and Gynaecology, Medical Faculty, Cologne University, 50931, Cologne, Germany. Electronic address:

The protocol of aseptic cryoprotectant-free vitrification on human spermatozoa is well documented. However, data about the effect of permeable cryoprotectants at this procedure is limited. Presented study aimed to test the aseptic capillary vitrification technologies using permeable cryoprotectant-included or cryoprotectant-free media. Thirty-two normal samples were included and analyzed after vitrification in three different media and thawing. Three treatment groups were formed: Group 1, basic medium; Group 2, basic medium with 0.25 M sucrose; Group 3, basic medium with glycerol. Before plunging into liquid nitrogen, capillaries were filled by 10 μl of spermatozoa suspension and isolated from liquid nitrogen by location in hermetically closed 0.25 ml straws. Progressive motility, plasma membrane integrity, total motility/viability after 24, 48 and 72 h in vitro culture, apoptosis and mitochondrial membrane potential (ΔΨm) were determined after thawing at 42 °C. Progressive motility of spermatozoa in groups 1, 2, 3 was 24.9 ± 1.7%, 34.5 ± 2.8% and 34.0 ± 1.4%, respectively (P<0.05). The plasma membrane integrity of spermatozoa in groups 2 and 3 (48.4 ± 2.9% and 45.5 ± 3.9%, respectively) was higher than in Group 1 (33.3 ± 2.1%, P < 0.05). After 24 h, 48 h and 72 h in vitro culture, the total motility and viability of spermatozoa in Group 1 was significantly lower than Group 2 and Group 3. The apoptosis rate in Group 3 (44.5 ± 3.0%) and Group 2 (47.7 ± 4.1%) were lower than in Group 1 (52.5 ± 4.4%; P < 0.05). ΔΨm rates in Group 3 and Group 2 were higher than in Group 1 (P < 0.05) with no statistical differences between this parameter in Group 2 and Group 3 (P > 0.1). In conclusion, supplementation of medium for aseptic capillary technology for cryoprotectant-free vitrification of human spermatozoa by permeable cryoprotectant does not improve the quality of spermatozoa after warming.
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http://dx.doi.org/10.1016/j.cryobiol.2021.01.006DOI Listing
April 2021

Vimentin 3 Expression in Prostate Cancer Cells.

Anticancer Res 2021 Jan;41(1):169-174

University Hospital of Cologne, Faculty of Medicine and University Hospital Cologne, Department of Urology, Cologne, Germany.

Background/aim: Vimentin3 (Vim3) was recently described as a tumour marker for the direct discrimination between benign and malignant kidney tumours. Here, we examined its expression in prostate cancer (PCa) cell lines and the regulation of its expression by endothelin receptors.

Materials And Methods: Prostate cancer cell lines (PC3, DU145, LNCap) were incubated with endothelin 1 (ET-1), BQ123 [endothelin A receptor (ETAR) antagonist], BQ788 [endothelin B receptor (ETBR) antagonist], BQ123+ET-1, BQ788+ET-1 for 24 h and a scratch assay was performed. Cell extracts were analysed by western blotting and qRT-PCR.

Results: ET-1 induced Vim3 overexpression. Blocking the ETBR in the different prostate cancer cell lines yielded a higher migration rate, whereby Vim3 expression was significantly increased.

Conclusion: Vim3 concentration increases in cell lines without a functional ETBR and may be used as a marker for PCas where ETBR is frequently methylated.
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http://dx.doi.org/10.21873/anticanres.14762DOI Listing
January 2021

Mxi-2 Dependent Regulation of p53 in Prostate Cancer.

Anticancer Res 2020 Oct;40(10):5539-5544

Department of Urology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany

Background/aim: Endothelin-1 (ET-1) is overexpressed in many types of cancer, inhibiting the release of the microRNA 15a (miR-15a) and inducing the production of Mxi-2. Our aim was to identify a molecular complex regulating p53 activity in prostate cancer (PCa).

Materials And Methods: DU145 cells were treated with ET-1, MAPK p38 inhibitor, Endothelin A receptor inhibitor (ETAR inhibitor) and Endothelin B receptor inhibitor (ETBR inhibitor). Extracts were analysed using Western Blot, immunoprecipitation and qRT-PCR. Furthermore, prostate cancer patient samples were analysed using qRT-PCR and ELISA.

Results: The hypothesised molecular complex was identified, with miR-15a, microRNA 1285 (miR-1285) and Mxi-2 levels up-regulated in patients in relation to increasing aggressiveness of PCa.

Conclusion: A complex composed of Argonaut 2 (Ago2)/Mxi-2/miR-1285 is involved in PCa. The expression of Mxi-2 correlates with increasing PCa aggressiveness and might be used as a non-invasive marker for the diagnosis and progression of PCa.
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http://dx.doi.org/10.21873/anticanres.14566DOI Listing
October 2020

Prediction of Radioresistant Prostate Cancer Based on Differentially Expressed Proteins.

Urol Int 2021 13;105(3-4):316-327. Epub 2020 Aug 13.

Department of Urology, University Hospital of Cologne, Cologne, Germany.

Introduction: Although relapses after radiotherapy are common in prostate cancer (PCA) patients, those with a high risk for radioresistance cannot be identified prior to treatment yet. Therefore, this proof-of-concept study was performed to compare protein expression profiles of patients with radio-recurrent PCA to patients treated with primary radical prostatectomy separated by Gleason risk groups. We hypothesized that radio-recurrent PCA have a similar protein expression as high-risk Gleason PCA.

Methods: Patient cohorts consisted of (i) 31 patients treated with salvage prostatectomy for locally recurrent PCA after primary radiotherapy and (ii) 94 patients treated with primary prostatectomy split into a Gleason high-risk (≥4 + 3; n = 42 [44.7%]) versus a low-risk group (≤3 + 4; n = 52 [55.3%]). Immunohistochemistry was performed using 15 antibodies with known association to radioresistance in PCA in vitro. ELISA was used for validation of selected markers in serum.

Results: Androgen receptor (AR) was overexpressed in most radio-recurrent PCA (89.7%) and in most primary high-risk Gleason PCA (87.8%; p = 0.851), while only 67.3% of the low-risk group showed an expression (p = 0.017). Considering the highest Gleason pattern in primary PCA, aldo-keto reductase family 1 member C3 (AKR1C3) was most similarly expressed by patients with radio-recurrent PCA and patients with Gleason patterns 4 and 5 (p = 0.827 and p = 0.893) compared to Gleason pattern 3 (p = 0.20). These findings were supported by ELISA.

Conclusion: This is the first study to evaluate protein markers in order to predict radioresistance in PCA. Our results point to AR and AKR1C3 as the most promising markers that might help stratify patients for radiotherapy.
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http://dx.doi.org/10.1159/000509447DOI Listing
July 2021

Vimentin 3 Allows Differentiation between Normozoospermia and Oligoasthenoteratozoospermia.

Dis Markers 2019 10;2019:9803498. Epub 2019 Dec 10.

Department of Urology, University Hospital of Cologne, Germany.

Vimentin is a structural protein predominantly located in the head of sperms. The function and localization of the previously identified truncated version, Vimentin 3 (Vim3), are still unknown. To investigate whether the expression of Vim3 can be used as a reliable marker for the differentiation of sperm quality, we analyzed ejaculates from patients with oligoasthenoteratozoospermia (OAT) syndrome and normozoospermia. We identified sperms with head, neck, and tail changes, which were less positive for Vim3 in OAT syndrome compared to normozoospermia. The expression of Vim3 was significantly downregulated in patients with OAT syndrome compared to sperms from patients with normozoospermia ( < 0.01). The ELISA analysis showed similar results as ejaculates from normozoospermic patients showed a significantly higher Vim3 concentration than patients with OAT syndrome ( < 0.001). This study demonstrates that Vim3 is more highly expressed in ejaculates from patients with normozoospermia compared to ejaculates from patients with OAT syndrome. Therefore, we postulate that Vim3 can be used to determine ejaculate quality. Furthermore, we identified the marker, Vim3, to differentiate between mature sperms with no morphological changes and sperms with head, neck, and tail changes. A lateral flow assay that allows quick analysis is currently under development.
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http://dx.doi.org/10.1155/2019/9803498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925920PMC
May 2020

Efficacy of the Oestrogen Antagonist Tamoxifen on Sperm Parameters in Patients with Idiopathic Oligoathenoteratozoospermia.

Urol Int 2019 8;103(1):108-115. Epub 2019 May 8.

Department of Urology, PAN-Clinic, Cologne, Germany.

Background: The oestrogen antagonist tamoxifen has been suggested as an empiric treatment option for treating idiopathic oligoathenoteratozoospermia (iOAT).

Objectives: To analyse the use of tamoxifen in iOAT.

Method: Fifty-seven men receiving tamoxifen for iOAT were recruited from 2016 to 2017 in a retrospective, single-centre setting. Hormone and semen analysis was performed before and after 3 months of treatment.

Results: After a 3-month treatment, serum levels of testosterone (3.4 ng/mL [2.7-4.8] vs. 5.3 [3.1-7.1]; p = 0.026), follicle stimulating hormone (FSH; 7.6 [5.9-11.5] vs. 15.9 mIU/mL [8.4-19.9]; p = 0.003) and luteinizing hormone (4.5 [3.3-6.6] vs. 7.6 mIU/mL [4.8-10.7]; p = 0.007) significantly increased. At a cut-off of >8.8 mIU/mL, serum levels of FSH were predictive for an improved sperm motility (OR 0.229 [95% CI 0.068-0.773]; p  = 0.018) and serum levels of inhibin B were predictive for an improved total sperm count at a cut-off of <82 ng/L (OR 18.0 [95% CI 1.267-255.744]; p = 0.033). During an 11 month-follow-up, patients receiving tamoxifen showed a clinical pregnancy rate of 42%, leading to a live birth rate of 56% of all pregnant women. Twenty-three per cent of all patients reported non-serious adverse events.

Conclusions: Tamoxifen is effective in improving the total sperm count as well as motility and can thus be safely used as empiric medical therapy in iOAT.
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http://dx.doi.org/10.1159/000500301DOI Listing
February 2020

Beyond the 3'UTR binding-microRNA-induced protein truncation via DNA binding.

Oncotarget 2018 Aug 28;9(67):32855-32867. Epub 2018 Aug 28.

Institute of Pathology, University Hospital of Cologne, Cologne, Germany.

Here, we present a miR mechanism which is active in the nucleus and is essential for the production of intron included, C-terminal truncated and biologically active proteins, like e.g. Vim3. We exemplified this mechanism by miRs, miR-15a and miR-498, which are overexpressed in clear cell renal carcinoma or oncocytoma. Both miRs directly interact with DNA in an intronic region, leading to transcriptional stop, and therefore repress the full length version of the pre-mRNA, resulting in intron included truncated proteins (Mxi-2 and Vim3). A computational survey shows that this miR:DNA interactions mechanism may be generally involved in regulating the human transcriptome, with putative interaction sites in intronic regions for over 1000 genes. In this work, an entirely new mechanism is revealed how miRs can repress full length protein translation, resulting in C-terminal truncated proteins.
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http://dx.doi.org/10.18632/oncotarget.26023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6132356PMC
August 2018

MicroRNAs as Urinary Biomarker for Oncocytoma.

Dis Markers 2018 16;2018:6979073. Epub 2018 Jul 16.

Institute of Pathology, University Hospital of Cologne, Kerpenerstraße 62, 50924 Cologne, Germany.

The identification of benign renal oncocytoma, its differentiation from malignant renal tumors, and their eosinophilic variants are a continuous challenge, influencing preoperative planning and being an unnecessary stress factor for patients. Regressive changes enhance the diagnostic dilemma, making evaluations by frozen sections or by immunohistology (on biopsies) unreliable. MicroRNAs (miRs) have been proposed as novel biomarkers to differentiate renal tumor subtypes. However, their value as a diagnostic biomarker of oncocytoma in urines based on mechanisms known in oncocytomas has not been exploited. We used urines from patients with renal tumors (oncocytoma, renal cell carcinoma: clear cell, papillary, chromophobe) and with other urogenital lesions. miRs were extracted and detected via qRT-PCR, the respective tumors analyzed by immunohistology. We found isocitrate dehydrogenase 2 upregulated in oncocytoma and oncocytic chromophobe carcinoma, indicating an increased Krebs cycle metabolism. Since we had shown that all renal tumors are stimulated by endothelin-1, we analyzed miRs preidentified by microarray after endothelin-1 stimulation of renal epithelial cells. Four miRs are proposed as urinary biomarkers due to their known regulatory mechanism in oncocytoma: miR-498 (formation of the oncocytoma-specific slice-form of vimentin, Vim3), miR-183 (associated with increased CO levels), miR-205, and miR-31 (signaling through downregulation of PKC epsilon, shown previously).
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http://dx.doi.org/10.1155/2018/6979073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6079495PMC
December 2018

Preoperative Stating of Pelvic Lymph Nodes in Prostate Cancer Patients Endorectal Magnetic Resonance Imaging.

Anticancer Res 2018 03;38(3):1763-1765

Department of Urology, Interbalkan Medical Center, Thessaloniki, Greece.

Background/aim: The aim of this study was to evaluate the diagnostic sensitivity, specificity and accuracy of endorectal magnetic resonance imaging (e-MRI), as a preoperative staging modality in the diagnosis of lymph node metastasis (LNM) in patients with prostate cancer (PCa).

Patients And Methods: Retrospectively, we analyzed data from N=168 patients who underwent radical prostatectomy (RP) between 2004 and 2013 at two tertiary medical centres. Prior to RP all patients underwent an e-MRI. Inclusion criteria were: PSA levels >20 ng/ml or Gleason score >7. Examinations were performed on a closed 1.0-T system combined with an endorectal body phased-array coil, imaging results were correlated with histopathology.

Results: 10.7% (N=18 patients) had histologically-proven LNM. e-MRI was true-positive in N=6 (33.3%) and false-negative N=12 patients (66.6%). N=150 (89.3%) patients without LNM e-MRI were true-negative in 96% and false-positive in 4%. Sensitivity was 96%, specificity was 33%, accuracy was 64.5%.

Conclusion: e-MRI can be considered a useful preoperative staging modality in diagnosis of LNM.
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http://dx.doi.org/10.21873/anticanres.12413DOI Listing
March 2018

Websites on Bladder Cancer: an Appropriate Source of Patient Information?

J Cancer Educ 2019 04;34(2):381-387

Department of Urology, University Hospital Cologne, Kerpener Straße 62, 50937, Cologne, Germany.

A growing number of patients search for health information online. An early investigation of websites about bladder cancer (BCa) revealed mostly incomplete and particularly inaccurate information. We analyzed the quality, readability, and popularity of the most frequented websites on BCa. An Internet search on www.google.com was performed for the term "bladder cancer." After selecting the most frequented websites for patient information, HONcode quality certification, Alexa popularity rank, and readability scores (according to US grade levels) were investigated. A 36-point checklist was used to assess the content according to the EAU guidelines on BCa, which was categorized into seven topics. The popularity of the 49 websites analyzed was average, with a median Alexa popularity rank of 41,698 (interquartile range [IQR] 7-4,671,246). The readability was rated difficult with 11 years of school education needed to understand the information. Thirteen (27%) websites were HONcode certified. Out of 343 topics (seven EAU guideline topics each on 49 websites), 79% were mentioned on the websites. Of these, 10% contained incorrect information, mostly outdated or biased, and 34% contained incomplete information. Publically provided websites mentioned more topics per website (median [IQR] 7 [5.5-7] vs. 5.5 [3.3-7]; p = 0.022) and showed less incorrect information (median [IQR] 0 [0-1] vs. 1 [0-1]; p = 0.039) than physician-provided websites. Our study revealed mostly correct but partially incomplete information on BCa websites for patients. Physicians and public organizations should strive to keep their website information up-to-date and unbiased to optimize patients' health literacy.
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http://dx.doi.org/10.1007/s13187-017-1316-2DOI Listing
April 2019

Immune Complex-Type Deposits in the Fischer-344 to Lewis Rat Model of Renal Transplantation and a Subset of Human Transplant Glomerulopathy.

Transplantation 2016 05;100(5):1004-14

1 Laboratory of Experimental Surgery, Department of General and Thoracic Surgery, Justus-Liebig-University Giessen, Giessen, Germany. 2 Institute of Pathology, University Hospital of Cologne, Cologne, Germany. 3 Institute of Transfusion Medicine, Hannover Medical School, Hannover, Germany. 4 Institute of Pathology, Department of Nephropathology, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany. 5 Institute of Pathology, Hannover Medical School, Hannover, Germany. 6 PATHOCOM, Rheine, Germany. 7 Department of General and Thoracic Surgery, Justus-Liebig-University Giessen, Giessen, Germany. 8 Medical Clinic III, University Hospital Hamburg-Eppendorf, Hamburg, Germany. 9 Department of General, Visceral and Cancer Surgery, Transplant Center Cologne, University of Cologne, Cologne, Germany. 10 Imperial College Kidney and Transplant Centre, Hammersmith Hospital, Imperial College Healthcare NHS Trust, London, United Kingdom. 11 Department of Cellular Pathology, Hammersmith Hospital, Imperial College Healthcare NHS Trust, London, United Kingdom.

Background: Antibody-mediated rejection is a leading cause for renal transplant loss. Rodent models are useful to dissect pathomechanisms and to develop treatment strategies. Although used for decades as a model, glomerular histopathological findings of Fischer-344 kidneys transplanted into Lewis rats have never been comprehensively described.

Methods: Kidneys from Fischer-344 rats were transplanted into Lewis rats as life-sustaining allografts without immunosuppression. Lewis isografts and normal Fischer-344 kidneys served as controls. Grafts were harvested at 9 days, 6 and 26 weeks. Histopathological examination included light microscopy, immunohistochemistry, and morphometry. Findings were compared with 51 human biopsies with transplant glomerulopathy.

Results: Most glomerular findings in rat allografts resembled human acute and chronic antibody-mediated rejection with glomerulitis, microthrombosis, microaneurysms, glomerular hypertrophy, podocyte loss, glomerular basement membrane splitting, and secondary focal and segmental glomerulosclerosis. In line with previous reports on nonendothelial antigens, glomerular immunoglobulin and C4d deposition was mostly nonendothelial. Only in 26-week allografts, we found mesangial and subendothelial immune complex-type electron-dense deposits. Similar deposits were found in 8 of 51 human biopsies with transplant glomerulopathy after rigorous exclusion of immune complexes of other cause, particularly recurrent glomerulonephritis and hepatitis C.

Conclusions: Thus, our model closely reflects the glomerular changes of acute antibody-mediated rejection in humans and of a special subset of human transplant glomerulopathy. The significance of alloimmune immune complex-type deposits in human transplants deserves further investigation.
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http://dx.doi.org/10.1097/TP.0000000000001068DOI Listing
May 2016

Vimentin 3, the new hope, differentiating RCC versus oncocytoma.

Dis Markers 2015 7;2015:368534. Epub 2015 Apr 7.

Institute of Pathology, University Hospital of Cologne, Kerpenerstraße 62, 50924 Cologne, Germany.

Vimentin is currently used to differentiate between malignant renal carcinomas and benign oncocytomas. Recent reports showing Vimentin positive oncocytomas seriously question the validity of this present diagnostic approach. Vimentin 3 is a spliced variant and ends with a unique C-terminal ending after exon 7 which differentiates it from the full length version that has 9 exons. Therefore, the protein size is different; the full length Vimentin version has a protein size of ~57 kDa and the truncated version of ~47 kDa. We designed an antibody, called Vim3, against the unique C-terminal ending of the Vimentin 3 variant. Using immune histology, immune fluorescence, Western blot, and qRT-PCR analysis, a Vim3 overexpression was detectable exclusively in oncocytoma, making the detection of Vim3 a potential specific marker for benign kidney tumors. This antibody is the first to clearly differentiate benign oncocytoma and the mimicking eosinophilic variants of the RCCs. This differentiation between malignant and benign RCCs is essential for operative planning, follow-up therapy, and patients' survival. In the future the usage of Vimentin antibodies in routine pathology has to be applied with care. Consideration must be given to Vimentin specific binding epitopes otherwise a misdiagnosis of the patients' tumor samples may result.
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http://dx.doi.org/10.1155/2015/368534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405285PMC
December 2015

ET-1 Induced Downregulation of MRP2 via miRNA 133a - A Marker for Tubular Nephrotoxicity?

Am J Nephrol 2015 8;41(3):191-9. Epub 2015 Apr 8.

Department of Pathology, University Hospital of Cologne, Cologne, Germany.

Background: Multiple drug resistance (MDR), known from treating malignant tumors with chemotherapy, increases the efflux of reabsorbed reagents in tumor cells. This mechanism has been reported in the renal proximal tubule and may prevent therapeutic tubular protection in proteinuria. Since endothelin-1 (ET-1), a major component in the urine of proteinuric patients, stimulates proximal tubules, its influence on MDR was analyzed with emphasis on the multidrug resistance-associated protein 2 (MRP2), a prominent transporter in the human proximal tubule and microRNA (miRNA) 133a.

Methods: ET-1 stimulated, cultured human renal proximal tubule cells (RPTECs), were analyzed via Western blot for the expression of MRP2 and via qRT-PCR for miRNA 133a. For direct interaction between the miRNA 133a and the 3'UTR of MRP2, an immunoprecipitation was performed using FITC-labelled miRNA 133a as capture, followed by MRP2 PCR analysis and Sanger sequencing. Murine Adriamycin nephropathic model and human proteinuric samples showed high levels of miRNA 133a but low levels of MRP2. The increasing miRNA 133a levels were detectable in urine samples of humans and animals.

Results: ET-1 activates the miRNA 133a, which can bind to the 3'UTR of MRP2 and is therefore responsible for the detectable decrease of MRP2.

Conclusion: This is the first report to analyze the correlation between ET-1-induced miRNA 133a overexpression in proteinuria resulting in MRP2 downregulation, which is a contributing factor for renal cytotoxicity. The detection of the miRNA 133a in urine samples can be possibly used as a monitor for cytotoxicity.
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http://dx.doi.org/10.1159/000381272DOI Listing
May 2016

MicroRNAs: Small but amazing, and their association with endothelin.

Life Sci 2012 Oct 4;91(13-14):475-89. Epub 2012 Jul 4.

Institute of Pathology, University Hospital, Kerpenerstraße 62, 50924 Koeln, Germany.

MicroRNAs (miRNAs) are small non-coding RNA molecules involved in the expressional regulation of genes by inhibiting gene translation. MicroRNAs are recruited and incorporated into the miRISC, ribonucleoprotein complex, targeting specific mRNAs through mechanisms specific for a miRNA sequence. Here we review the biogenesis, regulation, and monitoring of miRNAs, as well as the current evidence for potential roles of miRNAs in human diseases associated with activation of the endothelin system. These diseases include cancer, kidney disease, cardiovascular diseases, inflammatory diseases, infectious diseases, and blood diseases, that may all be aggravated by aberrant miRNA expression. In this review we will also discuss regulatory mechanisms determining production of miRNA as well as measuring or targeting miRNAs as potential novel approaches for diagnosis and treatment. Targeting miRNAs possibly will allow one to detect diseases or to interfere with the progression of diseases associated with activation of the endothelin system.
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http://dx.doi.org/10.1016/j.lfs.2012.06.025DOI Listing
October 2012

MicroRNA 15a, inversely correlated to PKCα, is a potential marker to differentiate between benign and malignant renal tumors in biopsy and urine samples.

Am J Pathol 2012 May 17;180(5):1787-97. Epub 2012 Mar 17.

Institute of Pathology, University Hospital, Cologne, Germany.

NF-κB signal transduction is a potential therapeutic target in many malignant tumors. We have recently shown, in malignant renal proximal tumor cells, that a transcription complex, consisting of NF-κB p65 and mitogen-activated protein kinase p38α, joined by protein kinase C (PKC) α, transmigrates into the nucleus. There, PKCα suppresses the nuclear release of primary microRNA (pri-miRNA) 15a. Induced by endothelin (ET)-1, a decrease in PKCα levels leads to increased miRNA 15a (miR-15A) expression. An identical system can be identified in renal carcinomas, in which, after nuclear transmigration, PKCα binds directly to pri-miRNA 15a in the nucleus. However, the pattern of PKC isoforms differs between malignant renal cell carcinoma (RCC) and benign oncocytoma. PKCα, a component of the transcription complex in tumors, is up-regulated in benign oncocytoma but down-regulated in RCC. Conversely, miRNA 15a is up-regulated in RCC and down-regulated in oncocytoma. A similar behavior is observed in chromophobe carcinoma, whereas differences are less pronounced in papillary RCC (type I): NF-κB target gene expression (ie, ET-1, ET-A and ET-B receptors, vascular cell adhesion molecule-1, IL-6, and fractalkine) is particularly high in malignant RCCs. Up-regulated miRNA 15a can be measured in urine from patients with RCC but is nearly undetectable in oncocytoma, other tumors, and urinary tract inflammation. Thus, the up-regulation of miRNA 15a may be an important marker to help identify malignant clear-cell RCCs in both biopsy and urine samples.
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http://dx.doi.org/10.1016/j.ajpath.2012.01.014DOI Listing
May 2012

Hepatocyte growth factor (HGF) inhibits collagen I and IV synthesis in hepatic stellate cells by miRNA-29 induction.

PLoS One 2011 9;6(9):e24568. Epub 2011 Sep 9.

Institute for Pathology, University Hospital Cologne, Cologne, Germany.

Background: In chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC.

Methodology: HSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3'-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting.

Principal Findings: The 3'-UTR of the collagen-1 and -4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-β exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis.

Conclusions: Upregulation of miRNA-29 by HGF and downregulation by TGF-β take part in the anti- or profibrogenic response of HSC, respectively.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024568PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3170366PMC
June 2012

Protein kinase C α regulates nuclear pri-microRNA 15a release as part of endothelin signaling.

Biochim Biophys Acta 2011 Oct 21;1813(10):1793-802. Epub 2011 Jun 21.

Institute of Pathology, University Hospital, Kerpenerstraße 62, 50924 Cologne, Germany.

Endothelin-1 induced signaling is characterized by an early induction of a nuclear factor-kappa B p65/mitogen-activated phosphokinase p38 transcription complex via its A-receptor versus a late induction via diacylglycerol, and protein kinase C. A possible interaction between these two pathways and a potential function for protein kinase C in this context has not previously been elucidated. Here we report that in Caki-1 tumor cells, protein kinase C α is a part of the transcription complex. With importin α4 and α5 as chaperones, the transcription complex transmigrates into the nucleus. Protein kinase C α blocks the nuclear release of pri-microRNA 15a by direct binding shown by electrophoretic mobility shift assay and Duolink immune histology. The expression levels of miRNA 15a can be further manipulated by transfection of si-protein kinase C α, or an expression vector containing protein kinase C α or miRNA 15. The miRNA 15a regulation by protein kinase C α is detectable in different malignant human tumor cell lines (renal cell carcinoma, breast carcinoma, and melanoma). Furthermore, all three cell lines harbor both endothelin receptors (ETAR/ETBR). Specific blockage of each receptor leads to major reduction of miRNA 15a expression due to increased nuclear protein kinase C α translocation. We conclude that the nuclear binding of pri-microRNA 15a is a novel function of protein kinase C α, which plays an important role in endothelin-1 mediated signaling. Since several endothelin-sensitive, malignant tumor cell lines harbor this regulation, it could indicate a more general role in tumor biology.
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http://dx.doi.org/10.1016/j.bbamcr.2011.06.006DOI Listing
October 2011

A p38-p65 transcription complex induced by endothelin-1 mediates signal transduction in cancer cells.

Biochim Biophys Acta 2008 Sep 16;1783(9):1613-22. Epub 2008 Apr 16.

Institute of Pathology, University Hospital, Kerpernerstrasse, 50931 Koeln, Germany.

Endothelin-1 is a powerful mitogen for various tumor and non-tumor cells. Its signaling cascade induces the inflammatory NF-kappaB complex, leading to expression of a number of target genes. In this context, MAPK p38 has been regarded as a potential phosphate donor for the p65 subunit of NF-kappaB. In the present study in HeLa cells, we have found that ET-1 induced signalling activates the NF-kappaB transcription complex (TC) in the nucleus at 6 h specifically via ET-A - but not ET-B receptor. The TC contains p65, p38 (alpha and beta) - binding to the NLS of p65 in the cytoplasm - as well as p50, but no IkappaBalpha. Specific p38 inhibition by SB203580 or by siRNA interferes markedly with gene expression of several target genes. Complex formation occurs in the cytoplasm, and both transcription factors transmigrate as a complex in the nucleus. Overexpression of p38, treatment with Chrysin, MG132, or dimethylformamide shows dependence of TC on p38 as partner. In other tumor cells lines studied, ET-1 activates TC, with p38 as an important complex partner of p65. TC-induction by ET-1 contains about twice the amount of p38 than by TNFalpha. Thus, p38 may be an additional therapeutic target to control inflammatory gene expression in tumor cells.
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http://dx.doi.org/10.1016/j.bbamcr.2008.04.003DOI Listing
September 2008
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