14 research outputs found
Handbook on effective asset recovery in compliance with European and international standards
No full textLes marqueurs de prolifération en oncologie vétérinaire : applications à l'étude pronostique du mastocytome cutané canin
No full textLe mastocytome cutané représente un type tumoral dominant de la cancérologie du chien mais demeure l'une des tumeurs canines au comportement clinique les plus imprévisibles. L'auteur évalue dans une étude rétrospective portant sur 120 chiens porteurs de mastocytome cutané, la valeur pronostique de la détection immunohistochimique des marqueurs de prolifération PCNA et Ki-67. Il montre que la détermination de l'expression de l'antigène Ki-67 permet de prédire avec une grande fiabilité l'évolution clinique post-chirurgicale de ce type tumoral
Cyclooxygenase-2 Expression in Bladder Cancer and Patient Prognosis: Results from a Large Clinical Cohort and Meta-Analysis
No full text27/02/14 meb. Open access paper, OK to pu
Transcriptome gene expression analysis of breast cancer using RNA-Seq
No full textabstract: Background: Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer deaths in females worldwide, accounting for 23% of all new cancer cases and 14% of all total cancer deaths in 2008. Five tumor-normal pairs of primary breast epithelial cells were treated for infinite proliferation by using a ROCK inhibitor and mouse feeder cells. Methods: Raw paired-end, 100x coverage RNA-Seq data was aligned to the Human Reference Genome Version 19 using BWA and Tophat. Gene differential expression analysis was completed using Cufflinks and Cuffdiff. Interactive Genome Viewer was used for data visualization. Results: 15 genes were found to be down-regulated by at least one log-fold change in 4/5 of tumor samples. 75 genes were found to be down-regulated in 3/5 of our tumor samples by at least one log-fold change. 11 genes were found to be up-regulated in 4/5 of our tumor samples, and 68 genes were identified to be up-regulated in 3/5 of the tumor samples by at least one-fold change. Conclusion: Expression changes in genes such as AZGP1, AGER, ALG11, and S1007 suggest a disruption in the glycosylation pathway. No correlation was found between Cufflink's Her2 gene-expression and DAKO score classification
Supplementary information files for "Firm carbon risk exposure and financial stability"
No full textSupplementary files for article "Firm carbon risk exposure and financial stability"This research investigates the influence of firm-level carbon risk on financial stability. Using a global sample of non-financial firms, we find that carbon risk negatively impacts financial stability. Financial constraints, corporate governance, and environmental innovation moderate this effect. The impact is stronger for firms with low R&D intensity and those in countries with high CO2 emissions, weak rule-of-law, low control of corruption, no carbon regulation, and low environmental regulation. Further tests show stronger effects in developing economies and in the post-global financial crisis and Paris Agreement periods. The findings highlight the need for tailored strategies to manage carbon risks.©The Author(s), CC-BY-NC-ND 4.0</p
PENDAMPINGNAN PENGAJUAN ARTIKEL ILMIAH DIJURNAL ILMIAH ONLINE BAGI MAHASISWA AKHIR STUDI
No full textThe purpose of this service is to provide student assistance in submitting and publishing articles in online journals and induction of the Mendeley application. The dedication method uses the Active Participant Participation (PRA) model. Submitting articles in scientific journals generally uses 5 steps to submit (according to the rules of the intended journal and depending on the OJS application used in the journal). After logging in to scientific journals (journals using OJS 2): Click new submission (1), Submit a draft article (Uploading the submission) (2), Fill in the metadata for each Author (Enter Metadata Submission) (3), Upload Supplementary Files namely uploading data related to the contents of the article for revision, generally there are separate instructions from the journal editor (4). Confirming Submission, The inside is the final step and saving your article file can be submitted. Organizing mentoring for submitting articles in online journals, induction of Mendeley applications, and providing expertise that makes it easier for students to write scientific articlesKata Kunci: Jurnal online; Artikel Ilmiah; Aplikasi mendele
Programmed death-ligand 1 expression influenced by tissue sample size. Scoring based on tissue microarrays’ and cross-validation with resections, in patients with, stage I–III, non-small cell lung carcinoma of the European Thoracic Oncology Platform Lungscape cohort
No full textPD-L1, as assessed by immunohistochemistry, is a predictive biomarker for immuno-oncology treatment in lung cancer. Different scoring methods have been used to assess its status, resulting in a wide range of positivity rates. We use the European Thoracic Oncology Platform Lungscape non-small cell lung carcinoma cohort to explore this issue. PD-L1 expression was assessed via immunohistochemistry on tissue microarrays (up to four cores per case), using the DAKO 28-8 immunohistochemistry assay, following a two-round external quality assessment procedure. All samples were analyzed under the same protocol. Cross-validation of scoring between tissue microarray and whole sections was performed in 10% randomly selected samples. Cutoff points considered: ≥1, 50 (primarily), and 25%. At the two external quality assessment rounds, tissue microarray scoring agreement rates between pathologists were: 73% and 81%. There were 2008 cases with valid immunohistochemistry tissue microarray results (50% all cores evaluable). Concordant cases at 1, 25, and 50% were: 85, 91, and 93%. Tissue microarray core results were identical for 70% of cases. Sensitivity of the tissue microarray method for 1, 25, and 50% was: 80, 78, and 79% (specificity: 90, 95, 98%). Complete agreement between tissue microarrays and whole sections was achieved for 60% of the cases. Highest sensitivity rates for 1% and 50% cutoffs were detected for higher number of cores. Underestimation of PD-L1 expression on small samples is more common than overestimation. We demonstrated that classification of PD-L1 on small biopsy samples does not represent the overall expression of PD-L1 in all non-small cell cancer carcinoma cases, although the majority of cases are ‘correctly’ classified. In future studies, sampling more and larger biopsies, recording the biopsy size and tumor load may permit further refinement, increasing predictive accuracy. © 2019, The Author(s), under exclusive licence to United States & Canadian Academy of Pathology
Author Correction: Federated learning enables big data for rare cancer boundary detection.
No full text10.1038/s41467-023-36188-7NATURE COMMUNICATIONS14
The Presence of Endometrial Cells in Peritoneal Fluid of Women With and Without Endometriosis
No full textTo reinforce Sampson's theory of retrograde menstruation in the pathogenesis of endometriosis, proof should be provided that during menstruation endometrial cells are present in peritoneal fluid (PF). We hypothesize that the prevalence of PF samples containing endometrial cells is higher in patients with endometriosis than in controls without endometriosis during menstruation. We selected from our biobank PF samples of 17 reproductive-age women with (n = 9) or without (n = 8) endometriosis who had received a diagnostic laparoscopy for investigation of pain/infertility. Peritoneal fluid had been collected during laparoscopy in the menstrual phase of the cycle, centrifuged, and the resulting pellet was stored at -80°C. About 5-μm sections of frozen PF pellets were stained using the Dako Envision Flex system with primary antibodies against epithelial cell adhesion molecule (Ep-CAM; endometrial epithelial cells), CD10 (endometrial stromal cells), prekeratin (epithelial/mesothelial cells), vimentin (endometrial/mesothelial/immune cells), calretinin (mesothelial cells), and CD68 (macrophages). The PF cells positive for Ep-CAM were detected in 5 of 9 patients with endometriosis and 6 of 8 controls (P = .62). CD10 stained positively in 6 of the 9 patients with endometriosis and 3 of the 8 controls (P = .35). Calretinin and prekeratin staining showed the presence of mesothelial cells in all pellets. Vimentin stained approximately 100% of the PF cells. CD68+ macrophages represented >50% of cells in all pellets. The prevalence of PF samples containing endometrial epithelial and stromal cells was not higher in patients with endometriosis than in controls without endometriosis during menstruation. Our findings question the relevance of endometrial cells in PF for the pathogenesis of endometriosis and support the importance of other mechanisms such as immune dysfunction and/or endometrial stem cells.sponsorship: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Funding of this research was obtained from the Research Foundation-Flanders (FWO, application numbers 11X5515N and 33915) and the Clinical Research Foundation of Leuven University Hospitals. (Research Foundation-Flanders (FWO)|11X5515N, Research Foundation-Flanders (FWO)|33915, Clinical Research Foundation of Leuven University Hospitals)status: Publishe
Detection of small intestinal IgA+TNFα+iNOS+ CELLS by Immunofluorescence Microscopy (IF)
No full textAuthors: Jennifer Gommerman & Jorg Fritz
### Abstract
The largest mucosal surface in the body is in the gastrointestinal (GI) tract, a location that is heavily colonized by normally harmless microbes. A key mechanism required for maintaining a homeostatic balance between this microbial burden and the lymphocytes that densely populate the GI tract is the production and trans-epithelial transport of poly-reactive IgA1. Within the mucosal tissues, B cells respond to cytokines, sometimes in the absence of T cell help, undergo class switch recombination (CSR) of their Immunoglobulin (Ig) receptors to IgA, and differentiate to become plasma cells (PC)2. However, IgA-secreting PC likely have additional attributes that are needed for coping with the tremendous bacterial load in the GI tract. We report that IgA+ PC also can produce the anti-microbial mediators TNFα and iNOS, which appear to arise in the unique environment of the gut, and may be critical to mount effective responses to microbial assault.
Here we described a detailed method to characterize and quantify IgA+iNOS+TNFα+ cells that we called TNFα-iNOS-producing (Tip)-PC in the lamina propria of mice by immunohistochemistry (IHC).
### Introduction
The largest mucosal surface in the body is in the gastrointestinal (GI) tract, a location that is heavily colonized by normally harmless microbes. A key mechanism required for maintaining a homeostatic balance between this microbial burden and the lymphocytes that densely populate the GI tract is the production and trans-epithelial transport of poly-reactive IgA1. Within the mucosal tissues, B cells respond to cytokines, sometimes in the absence of T cell help, undergo class switch recombination (CSR) of their Immunoglobulin (Ig) receptors to IgA, and differentiate to become plasma cells (PC)2. However, IgA-secreting PC likely have additional attributes that are needed for coping with the tremendous bacterial load in the GI tract. We report that IgA+ PC also can produce the anti-microbial mediators TNFα and iNOS, which appear to arise in the unique environment of the gut, and may be critical to mount effective responses to microbial assault.
Here we described a detailed method to characterize and quantify IgA+iNOS+TNFα+ cells that we called TNFα-iNOS-producing (Tip)-PC in the lamina propria of mice by immunohistochemistry (IHC).
### Reagents
1. Optimal Cutting Temperature (OCT) compound (Sakura Finetek)
- Base Molds (15×15 x 5 mm, Fisher Scientific)
- 2-Methyl-Butane (Fisher Scientific)
- Superfrost Plus Microscope Glass Slides and Coverslips (Fisher Scientific)
- Tween-20 (Sigma Aldrich)
- Tris-buffered saline (TBS)
- TBS-T (TBS plus 0.05% Tween-20)
- Phosphate buffered saline (PBS)
- normal rabbit serum (Jackson Laboratories)
- normal mouse serum (Jackson Laboratories),
- bovine serum albumin (BSA) (Sigma)
- rat anti-mouse CD16/CD32 (clone: 2.4G2, “Fc-block”)
-mDAPI (4’,6-diamidino-2-phenylindole) nucleic acid stain (InVitrogen)
- Gel/Mount (Biomeda Corporation)
- Dako Pen (Dako)
Antibodies:
1. rat anti-mouse IgA-FITC, Southern Biotechnologies 11-44-2
- rabbit anti-mouse iNOS-PE, Santa Cruz Biotechnology N20
- rat anti-mouse CD8α-PE eBioscience 53-6.7
- Anti-mouse monoclonal iNOS-ALEXA647 Santa Cruz Biotechnology C-11
- rat anti-mouse TNFα-PE eBioscience MP6-XT22
- rat anti-mouse TNFα-APC eBioscience MP6-XT22
- rat anti-mouse EpCAM-APC eBioscience G8.8
### Equipment
1. Leica CM3050 cryostat (Leica Microsystems)
- Leica DMRA2 microscope (Leica Microsystems)
- OpenLab imaging software (Improvision)
- Retiga EXi digital camera (Q Imaging)
- Photoshop CS5 (Adobe)
- Image J (NIH)
### Procedure
1. Cut the small intestines out of animals and remove the mesentery and fat.
- After gently pushing out the fecal content using a forceps, wash the small intestines gently twice in PBS and cut open longitudinally and in small pieces of approximately 1 cm using a scissor.
- Place tissue sections in base molds and cover with OCT compound and freeze in dry-ice-chilled 2-methyl-butane.
- Wrap frozen OCT-tissue blocks in aluminum paper and store at -80°C until used for sectioning.
- Cut intestinal tissue sections at 5 μm using a Leica CM3050 cryostat, mount on glass microscope slides, subsequently fix for seven minutes in ice-cold acetone and air-dry for one hour at room temperature. Fixed sections can be stored at -80°C in desiccant bags.
- Using a Dako Pen, mount hydrophobic barrier rings onto glass slides by circling tissues to limit reagent use for staining. By doing so approximately 400 μl of liquid (blocking or staining solutions) per slide is required.
- Rehydrate sections in TBS for 20 minutes followed by additional 20 minutes in TBS-T by placing them in slide-beakers applying gentle shaking.
- Block unspecific binding of antibodies by incubating tissue sections in TBS-T supplemented with 10% normal rabbit serum, 10% normal mouse serum, 5% sterile-filtered BSA and 2 mg/ml of a rat anti-mouse CD16/CD32 antibody for 30 minutes in a humidified chamber. After removal of the blocking solution, dip the slides briefly in TBS-T and then apply mixtures of the fluorochrome labeled antibodies diluted in TBS-T for 45 minutes in the dark in a humidified chamber.
- After removal of the staining solutions, wash the slides three times for ten minutes in TBS-T, once with TBS and once with PBS in slide-beakers applying gentle shaking.
- Stain the slides with DAPI nucleic acid stain for 30 seconds and wash three times with PBS in slide-beakers applying gentle shaking.
- Mount the slides with Gel/Mount medium putting coverslips.
- Acquire images with a Leica DMRA2 fluorescence microscope equipped with a Retiga EXi digital camera using OpenLab software. When comparing tissues of diverse genotypes, keep picture acquisition settings and exposure times for every fluorescent channel constant.
- Process images utilizing Photoshop CS5. When comparing images of diverse genotypes or comparing specific antibody staining with isotype controls, adjustments of input and output levels was applied for all compared images in an equal manner.
- Count the numbers of small intestinal LP IgA+ plasma cells and CD8α+ cells in a blinded fashion utilizing ImageJ. Analyse and quantify a total of five separate images from sections of six mice per group.
### Timing
Harvest tissue and preparation of sections will require approximately four hours. The staining of tissue sections will then take approximately five hours.
### Troubleshooting
1. We highly recommend to always apply a nucleic acid stain (DAPI) or staining intestinal epithelial cell s (EpCAM) as this will indicate you the quality of you tissue sections. Tissues depicting torn epithelial layers and/or disintegrated nuclei should be discarded.
- As some stains might be dim, we recommend to immediately acquire pictures after staining tissues. Avoid light exposure of stained tissues at all times.
- As staining intracellular antigens expressed at low levels can result in false positive results, we recommend to always include isotype control staining. If possible apply control staining utilizing tissues from TNFα and/or iNOS-deficient animals.
- Spinning the intracellular cocktail before you use it helps to minimize the background.
### Anticipated Results
Applying this staining methods we observes that IgA expression co-localizes with the expression of iNOS and TNFα with some, but not all IgA+ cells in the small intestinal LP tissue. Occasional co-expression of both iNOS and TNFα was observed in IgA+ cells, consistent with our flow cytometry data. Taken together, we have found that a proportion of IgA+ cells within the small intestinal LP express anti-microbial mediators TNFα and iNOS, while we do not detect significant expression of these molecules in other LP cells.
### References
1 Hooper, L. V. & Macpherson, A. J. Immune adaptations that maintain homeostasis with the intestinal microbiota. *Nat Rev Immunol* 10, 159-169, (2010).
- Fagarasan, S., Kawamoto, S., Kanagawa, O. & Suzuki, K. Adaptive immune regulation in the gut: T cell-dependent and T cell-independent IgA synthesis. *Annu Rev Immunol* 28, 243-273, (2010).
### Acknowledgements
We thank Dionne White in the Faculty of Medicine Flow Cytometry core facility. C.P. is supported by a CIHR operating grant MOP# 9862. R.C. is supported in part by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health. A.M. is supported by a CIHR operating grant MOP# 89783. J.H.F. acknowledges support by an APART-fellowship of the Austrian Academy of Sciences, McGill start-up funds and a CIHR operating grant MOP#114972. N.S. acknowledges the support of a CIHR Doctoral Award. J.L.G. is funded by the Canadian Institutes of Health Research (CIHR) and acknowledges the support of CIHR operating grant MOP# 67157 as well as infrastructure support from the Ontario Research Fund and that Canadian Foundation for Innovation.
### Figures
**Protocol with Figure 1.: Detection of TNF/iNOS/IgA in the small intestinal lamina propria by IF**
[Download Protocol with Figure 1](http://www.nature.com/protocolexchange/system/uploads/2002/original/iNOS_in_small_intestinal_lamina_propria.pdf?1321545660).
### Associated Publications
**Acquisition of a multifunctional IgA+ plasma cell phenotype in the gut**. Jörg H. Fritz, Olga Lucia Rojas, Nathalie Simard, Douglas D. McCarthy, Siegfried Hapfelmeier, Stephen Rubino, Susan J. Robertson, Mani Larijani, Jean Gosselin, Ivaylo I. Ivanov, Alberto Martin, Rafael Casellas, Dana J. Philpott, Stephen E. Girardin, Kathy D. McCoy, Andrew J. Macpherson, Christopher J. Paige, and Jennifer L. Gommerman. *Nature* [doi:10.1038/nature10698](http://dx.doi.org/10.1038/nature10698)
### Author information
**Jennifer Gommerman**, Gommerman Lab, University of Toronto
**Jorg Fritz**, McGill University
Correspondence to: Jorg Fritz ([email protected])
*Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2255) (2011) doi:10.1038/protex.2011.270. Originally published online 16 December 2011*
