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Article ; Online: Fibrosis and cancer: shared features and mechanisms suggest common targeted therapeutic approaches.

Landolt, Lea / Spagnoli, Giulio C / Hertig, Alexandre / Brocheriou, Isabelle / Marti, Hans-Peter

Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

2022 Volume 37, Issue 6, Page(s) 1024–1032

Abstract: Epidemiological studies support a strong link between organ fibrosis and epithelial cancers. Moreover, clinical and experimental investigations consistently indicate that these diseases intertwine and share strikingly overlapping features. As a ... ...

Abstract	Epidemiological studies support a strong link between organ fibrosis and epithelial cancers. Moreover, clinical and experimental investigations consistently indicate that these diseases intertwine and share strikingly overlapping features. As a deregulated response to injury occurring in all body tissues, fibrosis is characterized by activation of fibroblasts and immune cells, contributing to progressive deposition of extracellular matrix (ECM) and inflammation. Cancers are driven by genetic alterations resulting in dysregulated cell survival, proliferation and dissemination. However, non-cancerous components of tumour tissues including fibroblasts, inflammatory cells and ECM play key roles in oncogenesis and cancer progression by providing a pro-mutagenic environment where cancer cells can develop, favouring their survival, expansion and invasiveness. Additional commonalities of fibrosis and cancer are also represented by overproduction of growth factors, like transforming growth factor β, epithelial-to-mesenchymal transition, high oxidative stress, Hippo pathway dysfunctions and enhanced cellular senescence. Here, we review advances in the analysis of cellular and molecular mechanisms involved in the pathogenesis of both organ fibrosis and cancer, with particular reference to chronic kidney diseases and renal cell cancers. Most importantly, improved understanding of common features is contributing to the development of innovative treatment strategies targeting shared mechanisms.
MeSH term(s)	Epithelial-Mesenchymal Transition ; Extracellular Matrix ; Fibroblasts/metabolism ; Fibrosis ; Humans ; Neoplasms/etiology ; Neoplasms/metabolism ; Neoplasms/therapy
Language	English
Publishing date	2022-04-15
Publishing country	England
Document type	Journal Article ; Review ; Research Support, Non-U.S. Gov't
ZDB-ID	90594-x
ISSN	1460-2385 ; 0931-0509
ISSN (online)	1460-2385
ISSN	0931-0509
DOI	10.1093/ndt/gfaa301
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Article: MAGE-A10 Protein Expression in Advanced High Grade Serous Ovarian Cancer Is Associated with Resistance to First-Line Platinum-Based Chemotherapy.

Lisica Šikić, Nataša / Petrić Miše, Branka / Tomić, Snježana / Spagnol, Giulia / Matak, Luka / Juretić, Antonio / Spagnoli, Giulio

Cancers

2023 Volume 15, Issue 19

Abstract: Ovarian cancer has a dismal prognosis. Standard treatment following surgery relies on platinum-based chemotherapy. However, sizeable percentages of patients are unresponsive. Identification of markers predicting the response to chemotherapy might help ... ...

Abstract	Ovarian cancer has a dismal prognosis. Standard treatment following surgery relies on platinum-based chemotherapy. However, sizeable percentages of patients are unresponsive. Identification of markers predicting the response to chemotherapy might help select eligible patients and spare non-responding patients from treatment-associated toxicity. Cancer/testis antigens (CTAs) are expressed by healthy germ cells and malignant cells of diverse histological origin. This expression profile identifies them as attractive targets for cancer immunotherapies. We analyzed the correlations between expression of MAGE-A10 and New York esophageal-1 cancer (NY-ESO-1) CTAs at the protein level and the effectiveness of platinum-based chemotherapy in patients with advanced-stage high-grade serous ovarian carcinoma (HGSOC). MAGE-A10 and NY-ESO-1 protein expression was analyzed by immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded samples from 93 patients with advanced-stage HGSOC treated at our institutions between January 1996 and December 2013. The correlation between the expression of these markers and response to platinum-based chemotherapy, evaluated according to RECIST 1.1 criteria and platinum sensitivity, measured as platinum-free interval (PFI), progression free (PFS), and overall survival (OS) was explored. The MAGE-A10 protein expression predicted unresponsiveness to platinum-based chemotherapy (
Language	English
Publishing date	2023-09-23
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2527080-1
ISSN	2072-6694
ISSN	2072-6694
DOI	10.3390/cancers15194697
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Article ; Online: Prognostic Value of "Basal-like" Morphology, Tumor-Infiltrating Lymphocytes and Multi-MAGE-A Expression in Triple-Negative Breast Cancer.

Čeprnja, Toni / Tomić, Snježana / Perić Balja, Melita / Marušić, Zlatko / Blažićević, Valerija / Spagnoli, Giulio Cesare / Juretić, Antonio / Čapkun, Vesna / Vuger, Ana Tečić / Pogorelić, Zenon / Mrklić, Ivana

International journal of molecular sciences

2024 Volume 25, Issue 8

Abstract: Basal-like" (BL) morphology and the expression of cancer testis antigens (CTA) in breast cancer still have unclear prognostic significance. The aim of our research was to explore correlations of the morphological characteristics and tumor ... ...

Abstract	"Basal-like" (BL) morphology and the expression of cancer testis antigens (CTA) in breast cancer still have unclear prognostic significance. The aim of our research was to explore correlations of the morphological characteristics and tumor microenvironment in triple-negative breast carcinomas (TNBCs) with multi-MAGE-A CTA expression and to determine their prognostic significance. Clinical records of breast cancer patients who underwent surgery between January 2017 and December 2018 in four major Croatian clinical centers were analyzed. A total of 97 non-metastatic TNBCs with available tissue samples and treatment information were identified. Cancer tissue sections were additionally stained with programmed death-ligand 1 (PD-L1) Ventana (SP142) and multi-MAGE-A (mAb 57B). BL morphology was detected in 47 (49%) TNBCs and was associated with a higher Ki-67 proliferation index and histologic grade. Expression of multi-MAGE-A was observed in 77 (79%) TNBCs and was significantly associated with BL morphology. Lymphocyte-predominant breast cancer (LPBC) status was detected in 11 cases (11.3%) and significantly correlated with the Ki-67 proliferation index, increased number of intratumoral lymphocytes (itTIL), and PD-L1 expression. No impact of BL morphology, multi-MAGE-A expression, histologic type, or LPBC status on disease-free survival was observed. Our data suggest that tumor morphology could help identify patients with potential benefits from CTA-targeting immunotherapy.
MeSH term(s)	Adult ; Female ; Humans ; Antigens, Neoplasm/metabolism ; B7-H1 Antigen/metabolism ; Biomarkers, Tumor/metabolism ; Lymphocytes, Tumor-Infiltrating/metabolism ; Lymphocytes, Tumor-Infiltrating/immunology ; Lymphocytes, Tumor-Infiltrating/pathology ; Prognosis ; Triple Negative Breast Neoplasms/pathology ; Triple Negative Breast Neoplasms/metabolism ; Tumor Microenvironment
Chemical Substances	CD274 protein, human
Language	English
Publishing date	2024-04-20
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms25084513
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Article ; Online: Glomerular proteomic profiling reveals early differences between preexisting and de novo type 2 diabetes in human renal allografts.

Kipp, Anne / Marti, Hans-Peter / Babickova, Janka / Nakken, Sigrid / Leh, Sabine / Halden, Thea A S / Jenssen, Trond / Vikse, Bjørn Egil / Åsberg, Anders / Spagnoli, Giulio / Furriol, Jessica

BMC nephrology

2023 Volume 24, Issue 1, Page(s) 254

Abstract: Background: Diabetes mellitus (DM), either preexisting or developing after transplantation, remains a crucial clinical problem in kidney transplantation. To obtain insights into the molecular mechanisms underlying PTDM development and early glomerular ... ...

Abstract	Background: Diabetes mellitus (DM), either preexisting or developing after transplantation, remains a crucial clinical problem in kidney transplantation. To obtain insights into the molecular mechanisms underlying PTDM development and early glomerular damage before the development of histologically visible diabetic kidney disease, we comparatively analysed the proteome of histologically normal glomeruli from patients with PTDM and normoglycaemic (NG) transplant recipients. Moreover, to assess specificities inherent in PTDM, we also comparatively evaluated glomerular proteomes from transplant recipients with preexisting type 2 DM (T2DM). Methods: Protocol biopsies were obtained from adult NG, PTDM and T2DM patients one year after kidney transplantation. Biopsies were formalin-fixed and embedded in paraffin, and glomerular cross-sections were microdissected. A total of 4 NG, 7 PTDM and 6 T2DM kidney biopsies were used for the analysis. The proteome was determined by liquid chromatography-tandem mass spectrometry. Relative differences in protein abundance and significantly dysregulated pathways were analysed. Results: Proteins involved in cell adhesion, immune response, leukocyte transendothelial filtration, and cell localization and organization were less abundant in glomeruli from PTDM patients than in those from NG patients, and proteins associated with supramolecular fibre organization and protein-containing complex binding were more abundant in PTDM patients. Overall, proteins related to adherens and tight junctions and those related to the immune system, including leukocyte transendothelial migration, were more abundant in NG patients than in transplanted patients with DM, irrespective of the timing of its development. However, proteins included in cell‒cell junctions and adhesion, insulin resistance, and vesicle-mediated transport were all less abundant in PTDM patients than in T2DM patients. Conclusions: The glomerular proteome profile differentiates PTDM from NG and T2DM, suggesting specific pathogenetic mechanisms. Further studies are warranted to validate these results, potentially leading to an improved understanding of PTDM kidney transplant pathophysiology and to the identification of novel biomarkers.
MeSH term(s)	Adult ; Humans ; Kidney Transplantation ; Diabetes Mellitus, Type 2 ; Proteome ; Proteomics ; Kidney ; Diabetic Nephropathies ; Allografts
Chemical Substances	Proteome
Language	English
Publishing date	2023-08-25
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2041348-8
ISSN	1471-2369 ; 1471-2369
ISSN (online)	1471-2369
ISSN	1471-2369
DOI	10.1186/s12882-023-03294-z
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Article ; Online: High density of CXCL12-positive immune cell infiltration predicts chemosensitivity and recurrence-free survival in ovarian carcinoma.

Köhn, Philipp / Lalos, Alexandros / Posabella, Alberto / Wilhelm, Alexander / Tampakis, Athanasios / Caner, Ercan / Güth, Uwe / Stadlmann, Sylvia / Spagnoli, Giulio C / Piscuoglio, Salvatore / Richarz, Sabine / Delko, Tarik / Droeser, Raoul A / Singer, Gad

Journal of cancer research and clinical oncology

2023 Volume 149, Issue 20, Page(s) 17943–17955

Abstract: Background: Ovarian carcinoma is the most lethal gynecologic malignancy because of its late diagnosis, extremely high recurrence rate, and limited curative treatment options. In clinical practice, high-grade serous carcinoma (HGSC) predominates due to ... ...

Abstract	Background: Ovarian carcinoma is the most lethal gynecologic malignancy because of its late diagnosis, extremely high recurrence rate, and limited curative treatment options. In clinical practice, high-grade serous carcinoma (HGSC) predominates due to its frequency, high aggressiveness, and rapid development of drug resistance. Recent evidence suggests that CXCL12 is an important immunological factor in ovarian cancer progression. Therefore, we investigated the predictive and prognostic significance of the expression of this chemokine in tumor and immune cells in patients with HGSC. Methods: We studied a cohort of 47 primary high-grade serous ovarian carcinomas and their associated recurrences. A tissue microarray was constructed to evaluate the CXCL12 immunostained tumor tissue. CXCL12 expression was evaluated and statistically analyzed to correlate clinicopathologic data, overall survival, and recurrence-free survival. Results: A high proportion of CXCL12 + positive immune cells in primary ovarian serous carcinoma correlated significantly with chemosensitivity (p = 0.005), overall survival (p = 0.021), and longer recurrence-free survival (p = 0.038). In recurrent disease, high expression of CXCL12 was also correlated with better overall survival (p = 0.040). Univariate and multivariate analysis revealed that high CXCL12 + tumor-infiltrating immune cells (TICs) (HR 0.99, p = 0.042, HR 0.99, p = 0.023, respectively) and combined CXCL12 + /CD66b + infiltration (HR 0.15, p = 0.001, HR 0.13, p = 0.001, respectively) are independent favorable predictive markers for recurrence-free survival. Conclusion: A high density of CXCL12 + TICs predicts a good response to chemotherapy, leading to a better overall survival and a longer recurrence-free interval. Moreover, with concomitant high CXCL12/CD66b TIC density, it is an independent favorable predictor of recurrence-free survival in patients with ovarian carcinoma.
MeSH term(s)	Humans ; Female ; Ovarian Neoplasms/pathology ; Carcinoma, Ovarian Epithelial ; Prognosis ; Carcinoma ; Cystadenocarcinoma, Serous/pathology ; Biomarkers, Tumor/metabolism ; Chemokine CXCL12
Chemical Substances	Biomarkers, Tumor ; CXCL12 protein, human ; Chemokine CXCL12
Language	English
Publishing date	2023-11-15
Publishing country	Germany
Document type	Journal Article
ZDB-ID	134792-5
ISSN	1432-1335 ; 0171-5216 ; 0084-5353 ; 0943-9382
ISSN (online)	1432-1335
ISSN	0171-5216 ; 0084-5353 ; 0943-9382
DOI	10.1007/s00432-023-05466-8
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Article ; Online: Critical Evaluation of Transcripts and Long Noncoding RNA Expression Levels in Prostate Cancer Following Radical Prostatectomy.

Ruiz, Christian / Alborelli, Ilaria / Manzo, Massimilinao / Calgua, Byron / Keller, Eveline Barbara / Vuaroqueaux, Vincent / Quagliata, Luca / Rentsch, Cyrill A / Spagnoli, Giulio Cesare / Diener, Pierre André / Bubendorf, Lukas / Morant, Rudolf / Eppenberger-Castori, Serenella

Pathobiology : journal of immunopathology, molecular and cellular biology

2023 Volume 90, Issue 6, Page(s) 400–408

Abstract: Introduction: The clinical course of prostate cancer (PCa) is highly variable, ranging from indolent behavior to rapid metastatic progression. The Gleason score is widely accepted as the primary histologic assessment tool with significant prognostic ... ...

Abstract	Introduction: The clinical course of prostate cancer (PCa) is highly variable, ranging from indolent behavior to rapid metastatic progression. The Gleason score is widely accepted as the primary histologic assessment tool with significant prognostic value. However, additional biomarkers are required to better stratify patients, particularly those at intermediate risk. Methods: In this study, we analyzed the expression of 86 cancer hallmark genes in 171 patients with PCa who underwent radical prostatectomy and focused on the outcome of the 137 patients with postoperative R0-PSA0 status. Results: Low expression of the IGF1 and SRD52A, and high expression of TIMP2, PLAUR, S100A2, and CANX genes were associated with biochemical recurrence (BR), defined as an increase of prostate-specific antigen above 0.2 ng/mL. Furthermore, the analysis of the expression of 462 noncoding RNAs (ncRNA) in a sub-cohort of 39 patients with Gleason score 7 tumors revealed that high levels of expression of the ncRNAs LINC00624, LINC00593, LINC00482, and cd27-AS1 were significantly associated with BR. Our findings provide further evidence for tumor-promoting roles of ncRNAs in PCa patients at intermediate risk. The strong correlation between expression of LINC00624 and KRT8 gene, encoding a well-known cell surface protein present in PCa, further supports a potential contribution of this ncRNA to PCa progression. Conclusion: While larger and further studies are needed to define the role of these genes/ncRNA in PCa, our findings pave the way toward the identification of a subgroup of patients at intermediate risk who may benefit from adjuvant treatments and new therapeutic agents.
MeSH term(s)	Male ; Humans ; RNA, Long Noncoding/genetics ; Prostatic Neoplasms/genetics ; Prostatic Neoplasms/surgery ; Prostatic Neoplasms/pathology ; Prostate/pathology ; Prostate-Specific Antigen ; Prostatectomy ; Neoplasm Recurrence, Local/genetics ; Neoplasm Recurrence, Local/surgery ; Neoplasm Grading
Chemical Substances	RNA, Long Noncoding ; Prostate-Specific Antigen (EC 3.4.21.77)
Language	English
Publishing date	2023-07-18
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	1022703-9
ISSN	1423-0291 ; 1015-2008
ISSN (online)	1423-0291
ISSN	1015-2008
DOI	10.1159/000531175
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Article ; Online: Assessing Autophagy During Retinoid Treatment of Breast Cancer Cells.

Parejo, Sarah / Tschan, Mario P / Muraro, Manuele G / Garattini, Enrico / Spagnoli, Giulio C / Schläfli, Anna M

Methods in molecular biology (Clifton, N.J.)

2019 Volume 2019, Page(s) 237–256

Abstract: Retinoids are derived from vitamin A through a multi-step process. Within a target cell, retinoids regulate gene expression by activating the retinoid acid receptors (RAR) and retinoid x receptors (RXR), which are ligand-dependent transcription factors. ... ...

Abstract	Retinoids are derived from vitamin A through a multi-step process. Within a target cell, retinoids regulate gene expression by activating the retinoid acid receptors (RAR) and retinoid x receptors (RXR), which are ligand-dependent transcription factors. Besides its therapeutic use in dermatological disorders, all-trans retinoic acid (ATRA) is successfully utilized to treat acute promyelocytic leukemia (APL) patients. The use of ATRA in APL patients is the first example of clinically useful differentiation therapy. Therapeutic strategies aiming at cancer cell differentiation have great potential for solid tumors, including breast cancer. The few clinical studies conducted with ATRA in breast cancer are rather disappointing. However, these studies did not take into account the heterogeneity of the disease and were conducted on unselected cohorts of patients.We recently showed that ATRA treatment of breast cancer cells induces autophagy, a highly conserved process aiming at degrading and recycling superfluous or harmful cellular components. In addition, autophagy inhibition significantly increases the therapeutic activity of ATRA. This finding is of fundamental importance, since autophagy has a dual role in cancer. Whereas autophagy may be a protective mechanism during the initial phases of cancer development, it may support cancer cell survival in already established tumors. Furthermore, autophagy can lower or enhance therapeutic efficiency, depending on the tumor type and the anticancer agent considered. Therefore, it is important to investigate the role of autophagy in the context of specific tumors and therapeutic approaches. Accurate autophagy studies are challenging given the dynamic nature of the process and the difficulty of measuring the rate of autophagosome degradation (autophagic flux). In this chapter, we provide protocols for a careful assessment of the autophagic flux in ATRA treated 2D and 3D breast cancer cultures.
MeSH term(s)	Antineoplastic Agents/pharmacology ; Autophagy/drug effects ; Breast Neoplasms/drug therapy ; Breast Neoplasms/metabolism ; Cell Culture Techniques ; Cell Line, Tumor ; Cell Separation ; Female ; Flow Cytometry ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Tretinoin/pharmacology
Chemical Substances	Antineoplastic Agents ; Tretinoin (5688UTC01R)
Language	English
Publishing date	2019-07-29
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-9585-1_17
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Article: Prognostic Significance of Lymphocyte Infiltrate Localization in Triple-Negative Breast Cancer.

Čeprnja, Toni / Mrklić, Ivana / Perić Balja, Melita / Marušić, Zlatko / Blažićević, Valerija / Spagnoli, Giulio Cesare / Juretić, Antonio / Čapkun, Vesna / Tečić Vuger, Ana / Vrdoljak, Eduard / Tomić, Snježana

Journal of personalized medicine

2022 Volume 12, Issue 6

Abstract: High infiltration by tumor-infiltrating lymphocytes (TILs) is associated with favorable prognosis in different tumor types, but the clinical significance of their spatial localization within the tumor microenvironment is debated. To address this issue, ... ...

Abstract	High infiltration by tumor-infiltrating lymphocytes (TILs) is associated with favorable prognosis in different tumor types, but the clinical significance of their spatial localization within the tumor microenvironment is debated. To address this issue, we evaluated the accumulation of intratumoral TILs (itTILs) and stromal TILs (sTILs) in samples from 97 patients with early triple-negative breast cancer (TNBC) in the center (sTIL central) and periphery (sTIL peripheral) of tumor tissues. Moreover, the presence of primary and secondary lymphoid aggregates (LAs) and the expression levels of the cancer testis antigen (CTA), NY-ESO-1, and PD-L1 were explored. High infiltration by itTILs was observed in 12/97 samples (12.3%), unrelated to age, Ki67 expression, tumor size, histologic type and grade, and LA presence. NY-ESO-1 was expressed in tumor cells in 37 samples (38%), with a trend suggesting a correlation with itTIL infiltration (p = 0.0531). PD-L1 expression was detected in immune cells in 47 samples (49%) and was correlated with histologic grade, sTILs, and LA formation. The presence of primary LAs was significantly correlated with better disease-free survival (DFS) (p = 0.027). Moreover, no tumor progression was observed during >40 months of clinical follow up in the 12 patients with high itTILs or in the 14 patients with secondary LAs. Thus, careful evaluation of lymphoid infiltrate intratumoral localization might provide important prognostic information.
Language	English
Publishing date	2022-06-08
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2662248-8
ISSN	2075-4426
ISSN	2075-4426
DOI	10.3390/jpm12060941
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Article: High Density of CD16+ Tumor-Infiltrating Immune Cells in Recurrent Ovarian Cancer Is Associated with Enhanced Responsiveness to Chemotherapy and Prolonged Overall Survival.

Lalos, Alexandros / Neri, Ornella / Ercan, Caner / Wilhelm, Alexander / Staubli, Sebastian / Posabella, Alberto / Weixler, Benjamin / Terracciano, Luigi / Piscuoglio, Salvatore / Stadlmann, Sylvia / Spagnoli, Giulio C / Droeser, Raoul A / Singer, Gad

Cancers

2021 Volume 13, Issue 22

Abstract: Background: Ovarian cancer (OC) is the most aggressive and fatal malignancy of the female reproductive system. Debulking surgery with adjuvant chemotherapy represents the standard treatment, but recurrence rates are particularly high. Over the past ... ...

Abstract	Background: Ovarian cancer (OC) is the most aggressive and fatal malignancy of the female reproductive system. Debulking surgery with adjuvant chemotherapy represents the standard treatment, but recurrence rates are particularly high. Over the past decades, the association between the immune system and cancer progression has been extensively investigated. However, the interaction between chemotherapy and cancer immune infiltration is still unclear. In this study, we examined the prognostic role of CD16 expression in OC, as related to the effectiveness of standard adjuvant chemotherapy treatment. Methods: We analyzed the infiltration by immune cells expressing CD16, a well-characterized natural killer (NK) and myeloid cell marker, in a tissue microarray (TMA) of 47 patient specimens of primary OCs and their matching recurrences by immunohistochemistry (IHC). We analyzed our data first in the whole cohort, then in the primary tumors, and finally in recurrences. We focused on recurrence-free survival (RFS), overall survival (OS), and chemosensitivity. Chemosensitivity was defined as RFS of more than 6 months. Results: There was no significant correlation between CD16 expression and prognosis in primary carcinomas. However, interestingly, a high density of CD16-expressing tumor-infiltrating immune cells (TICs) in recurrent carcinoma was associated with better RFS ( Conclusions: The high density of CD16-expressing TICs in recurrent ovarian cancer is associated with a better RFS and OS, thereby suggesting a previously unsuspected interaction between standard OC chemotherapy and immune cell infiltration.
Language	English
Publishing date	2021-11-18
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2527080-1
ISSN	2072-6694
ISSN	2072-6694
DOI	10.3390/cancers13225783
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Article: T cell epitope-mapping by cytokine gene expression assay.

Provenzano, Maurizio / Spagnoli, Giulio C

Methods in molecular biology (Clifton, N.J.)

2009 Volume 514, Page(s) 107–118

Abstract: The following method describes the identification of candidate immunogenic peptides through their ability to recall an immune T-cell activation from peripheral blood mononuclear cells (PBMCs) of individuals with defined HLA-peptide restrictions that have ...

Abstract	The following method describes the identification of candidate immunogenic peptides through their ability to recall an immune T-cell activation from peripheral blood mononuclear cells (PBMCs) of individuals with defined HLA-peptide restrictions that have been previously exposed to the antigen. Isolated PBMCs are plated out at a concentration of 1 x 10(6) cells/ml in a 200 microl medium and incubated overnight to reduce cytokine gene expression due to cell manipulation. After starving, cells are either directly stimulated with individual peptides or not stimulated and incubated from 3 to 12 h according to experimental conditions. Quantitative real-time PCR (qrt-PCR) is performed on reverse-transcribed complementary DNA (cDNA) from total RNA that is isolated from peptide-cultured PBMCs. To perform high quality qrt-PCR, primers and probes are designed to span exon-intron junctions in order to prevent amplification of genomic DNA and to produce amplicons <150 base pairs (bp). Real-time monitoring of fluorescent emission from the cleavage of sequence specific probe by the nuclease activity of Taq polymerase (TaqMan method) defines threshold cycles during exponential phases of amplification. Standard curves of copy numbers of the gene of interest are normalized using as reference copy numbers of control genes.
MeSH term(s)	Actins/metabolism ; Cytokines/genetics ; DNA/genetics ; DNA Primers/metabolism ; Epitope Mapping/methods ; Epitopes, T-Lymphocyte/analysis ; Epitopes, T-Lymphocyte/immunology ; Epitopes, T-Lymphocyte/pharmacology ; Gene Dosage ; Gene Expression Profiling ; Gene Expression Regulation/drug effects ; Gene Expression Regulation/immunology ; Genome, Human ; Humans ; Peptides ; RNA/genetics ; Reference Standards ; Reverse Transcriptase Polymerase Chain Reaction ; T-Lymphocytes/drug effects ; T-Lymphocytes/immunology ; T-Lymphocytes/metabolism
Chemical Substances	Actins ; Cytokines ; DNA Primers ; Epitopes, T-Lymphocyte ; Peptides ; RNA (63231-63-0) ; DNA (9007-49-2)
Language	English
Publishing date	2009
Publishing country	United States
Document type	Journal Article
ISSN	1064-3745
ISSN	1064-3745
DOI	10.1007/978-1-60327-527-9_8
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