| 1. |
- O'Leary, Patrick C., et al.
(author)
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Peroxiredoxin-1 protects estrogen receptor alpha from oxidative stress-induced suppression and is a protein biomarker of favorable prognosis in breast cancer
- 2014
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In: Breast Cancer Research. - : Springer Science and Business Media LLC. - 1465-5411 .- 1465-542X. ; 16:4, s. R79-
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Journal article (peer-reviewed)abstract
- Introduction: Peroxiredoxin-1 (PRDX1) is a multifunctional protein, acting as a hydrogen peroxide (H2O2) scavenger, molecular chaperone and immune modulator. Although differential PRDX1 expression has been described in many tumors, the potential role of PRDX1 in breast cancer remains highly ambiguous. Using a comprehensive antibody-based proteomics approach, we interrogated PRDX1 protein as a putative biomarker in estrogen receptor (ER)-positive breast cancer. Methods: An anti-PRDX1 antibody was validated in breast cancer cell lines using immunoblotting, immunohistochemistry and reverse phase protein array (RPPA) technology. PRDX1 protein expression was evaluated in two independent breast cancer cohorts, represented on a screening RPPA (n = 712) and a validation tissue microarray (n = 498). In vitro assays were performed exploring the functional contribution of PRDX1, with oxidative stress conditions mimicked via treatment with H2O2, peroxynitrite, or adenanthin, a PRDX1/2 inhibitor. Results: In ER-positive cases, high PRDX1 protein expression is a biomarker of improved prognosis across both cohorts. In the validation cohort, high PRDX1 expression was an independent predictor of improved relapse-free survival (hazard ratio (HR) = 0.62, 95% confidence interval (CI) = 0.40 to 0.96, P = 0.032), breast cancer-specific survival (HR = 0.44, 95% CI = 0.24 to 0.79, P = 0.006) and overall survival (HR = 0.61, 95% CI = 0.44 to 0.85, P = 0.004). RPPA screening of cancer signaling proteins showed that ER alpha protein was upregulated in PRDX1 high tumors. Exogenous H2O2 treatment decreased ER alpha protein levels in ER-positive cells. PRDX1 knockdown further sensitized cells to H2O2- and peroxynitrite-mediated effects, whilst PRDX1 overexpression protected against this response. Inhibition of PRDX1/2 antioxidant activity with adenanthin dramatically reduced ER alpha levels in breast cancer cells. Conclusions: PRDX1 is shown to be an independent predictor of improved outcomes in ER-positive breast cancer. Through its antioxidant function, PRDX1 may prevent oxidative stress-mediated ER alpha loss, thereby potentially contributing to maintenance of an ER-positive phenotype in mammary tumors. These results for the first time imply a close connection between biological activity of PRDX1 and regulation of estrogen-mediated signaling in breast cancer.
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| 2. |
- Burdzinska, Anna, et al.
(author)
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Intraurethral co-transplantation of bone marrow mesenchymal stem cells and muscle-derived cells improves the urethral closure
- 2018
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In: Stem Cell Research and Therapy. - : Springer Science and Business Media LLC. - 1757-6512. ; 9:1
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Journal article (peer-reviewed)abstract
- Background: Cell therapy constitutes an attractive alternative to treat stress urinary incontinence. Although promising results have been demonstrated in this field, the procedure requires further optimization. The most commonly proposed cell types for intraurethral injections are muscle derived cells (MDCs) and mesenchymal stem/stromal cell (MSCs). The aim of this study was to evaluate the effects of MDC-MSC co-transplantation into the urethra. Methods: Autologous transplantation of labeled MDCs, bone marrow MSCs or co-transplantation of MDC-MSC were performed in aged multiparous female goats (n = 6 in each group). The mean number of cells injected per animal was 29.6 × 106(± 4.3 × 106). PBS-injected animals constituted the control group (n = 5). Each animal underwent urethral pressure profile (UPP) measurements before and after the injection procedure. The maximal urethral closure pressure (MUCP) and functional area (FA) of UPPs were calculated. The urethras were collected at the 28th or the 84th day after transplantation. The marker fluorochrome (DID) was visualized and quantified using in vivo imaging system in whole explants. Myogenic differentiation of the graft was immunohistochemically evaluated. Results: The grafted cells were identified in all urethras collected at day 28 regardless of injected cell type. At this time point the strongest DID-derived signal (normalized to the number of injected cells) was noted in the co-transplanted group. There was a distinct decline in signal intensity between day 28 and day 84 in all types of transplantation. Both MSCs and MDCs contributed to striated muscle formation if transplanted directly to the external urethral sphincter. In the MSC group those events were rare. If cells were injected into the submucosal region they remained undifferentiated usually packed in clearly distinguishable depots. The mean increase in MUCP after transplantation in comparison to the pre-transplantation state in the MDC, MSC and MDC-MSC groups was 12.3% (± 11.2%, not significant (ns)), 8.2% (± 9.6%, ns) and 24.1% (± 3.1%, p = 0.02), respectively. The mean increase in FA after transplantation in the MDC, MSC and MDC-MSC groups amounted to 17.8% (± 15.4%, ns), 15.2% (± 12.9%, ns) and 17.8% (± 2.5%, p = 0.04), respectively. Conclusions: The results suggest that MDC-MSC co-transplantation provides a greater chance of improvement in urethral closure than transplantation of each population alone.
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| 3. |
- Oei, Vincent Yi Sheng, et al.
(author)
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Intrinsic Functional Potential of NK-Cell Subsets Constrains Retargeting Driven by Chimeric Antigen Receptors
- 2018
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In: CANCER IMMUNOLOGY RESEARCH. - : AMER ASSOC CANCER RESEARCH. - 2326-6066 .- 2326-6074. ; 6:4, s. 467-480
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Journal article (peer-reviewed)abstract
- Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19(+) targets, and maintained their intrinsic degranulation response toward CD19(-) K562 cells. The response of redirected NK-cell subsets against CD19(+) targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA-specific KIR(s), displayed superior ability to kill CD19(+), HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells.
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| 4. |
- O'Leary, Patrick C., et al.
(author)
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Systematic antibody generation and validation via tissue microarray technology leading to identification of a novel protein prognostic panel in breast cancer
- 2013
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In: BMC Cancer. - : Springer Science and Business Media LLC. - 1471-2407. ; 13
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Journal article (peer-reviewed)abstract
- Background: Although omic-based discovery approaches can provide powerful tools for biomarker identification, several reservations have been raised regarding the clinical applicability of gene expression studies, such as their prohibitive cost. However, the limited availability of antibodies is a key barrier to the development of a lower cost alternative, namely a discrete collection of immunohistochemistry (IHC)-based biomarkers. The aim of this study was to use a systematic approach to generate and screen affinity-purified, mono-specific antibodies targeting progression-related biomarkers, with a view towards developing a clinically applicable IHC-based prognostic biomarker panel for breast cancer. Methods: We examined both in-house and publicly available breast cancer DNA microarray datasets relating to invasion and metastasis, thus identifying a cohort of candidate progression-associated biomarkers. Of these, 18 antibodies were released for extended analysis. Validated antibodies were screened against a tissue microarray (TMA) constructed from a cohort of consecutive breast cancer cases (n = 512) to test the immunohistochemical surrogate signature. Results: Antibody screening revealed 3 candidate prognostic markers: the cell cycle regulator, Anillin (ANLN); the mitogen-activated protein kinase, PDZ-Binding Kinase (PBK); and the estrogen response gene, PDZ-Domain Containing 1 (PDZK1). Increased expression of ANLN and PBK was associated with poor prognosis, whilst increased expression of PDZK1 was associated with good prognosis. A 3-marker signature comprised of high PBK, high ANLN and low PDZK1 expression was associated with decreased recurrence-free survival (p < 0.001) and breast cancer-specific survival (BCSS) (p < 0.001). This novel signature was associated with high tumour grade (p < 0.001), positive nodal status (p = 0.029), ER-negativity (p = 0.006), Her2-positivity (p = 0.036) and high Ki67 status (p < 0.001). However, multivariate Cox regression demonstrated that the signature was not a significant predictor of BCSS (HR = 6.38; 95% CI = 0.79-51.26, p = 0.082). Conclusions: We have developed a comprehensive biomarker pathway that extends from discovery through to validation on a TMA platform. This proof-of-concept study has resulted in the identification of a novel 3-protein prognostic panel. Additional biochemical markers, interrogated using this high-throughput platform, may further augment the prognostic accuracy of this panel to a point that may allow implementation into routine clinical practice.
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| 5. |
- Zarychta-Wiśniewska, Weronika, et al.
(author)
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In vivo imaging system for explants analysis—A new approach for assessment of cell transplantation effects in large animal models
- 2017
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:9
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Journal article (peer-reviewed)abstract
- Introduction: Despite spectacular progress in cellular transplantology, there are still many concerns about the fate of transplanted cells. More preclinical studies are needed, especially on large animal models, to bridge the translational gap between basic research and the clinic. Herein, we propose a novel approach in analysis of cell transplantation effects in large animals explants using in vivo imaging system (IVIS®) or similar equipment. Material and methods: In the in vitro experiment cells labeled with fluorescent membrane dyes: DID (far red) or PKH26 (orange) were visualized with IVIS®. The correlation between the fluorescence signal and cell number with or without addition of minced muscle tissue was calculated. In the ex vivo study urethras obtained from goats after intraurethral cells (n = 9) or PBS (n = 4) injections were divided into 0.5 cm cross-slices and analyzed by using IVIS®. Automatic algorithm followed or not by manual setup was used to separate specific dye signal from tissue autofluorescence. The results were verified by systematic microscopic analysis of standard 10 μm specimens prepared from slices before and after immunohistochemical staining. Comparison of obtained data was performed using diagnostic test function. Results: Fluorescence signal strength in IVIS® was directly proportional to the number of cells regardless of the dye used and detectable for minimum 0.25x106 of cells. DID-derived signal was much less affected by the background signal in comparison to PKH26 in in vitro test. Using the IVIS® to scan explants in defined arrangement resulted in precise localization of DID but not PKH26 positive spots. Microscopic analysis of histological specimens confirmed the specificity (89%) and sensitivity (80%) of IVIS® assessment relative to DID dye. The procedure enabled successful immunohistochemical staining of specimens derived from analyzed slices. Conclusions: The IVIS® system under appropriate conditions of visualization and analysis can be used as a method for ex vivo evaluation of cell transplantation effects. Presented protocol allows for evaluation of cell delivery precision rate, enables semi-quantitative assessment of signal, preselects material for further analysis without interfering with the tissue properties. Far red dyes are appropriate fluorophores to cell labeling for this application.
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