125 results on '"Usal C"'
Search Results
2. Indirect CD4+ TH1 Response, Antidonor Antibodies and Diffuse C4d Graft Deposits in Long-Term Recipients Conditioned by Donor Antigens Priming
- Author
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Ballet, C., Renaudin, K., Degauque, N., Mai, H.L., Boëffard, F., Lair, D., Berthelot, L., Feng, C., Smit, H., Usal, C., Heslan, M., Josien, R., Brouard, S., and Soulillou, J.-P.
- Published
- 2009
- Full Text
- View/download PDF
3. New lines of GFP transgenic rats relevant for regenerative medicine and gene therapy
- Author
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Remy, S., Tesson, L., Usal, C., Menoret, S., Bonnamain, V., Nerriere-Daguin, V., Rossignol, J., Boyer, C., Nguyen, T. H., Naveilhan, P., Lescaudron, L., and Anegon, I.
- Published
- 2010
- Full Text
- View/download PDF
4. Generation and Characterization of IgM, Rag1 and/or ILR2g knockout Immunodeficient Rats.: Abstract# D2797
- Author
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Menoret, S., Usal, C., Tesson, L., Remy, S., Thinard, R., de Cian, A., Ouisse, L., Fraichard, A., Buelow, R., Concordet, J., Giovannangeli, C., and Anegon, I.
- Published
- 2014
5. Tranilast, an Analogue of a Tryptophan Catabolite, Induces Allograft Tolerance By Myeloid-Derived Suppressor Cells (MDSCs).: Abstract# 1417
- Author
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Menoret, S., Bezie, S., Li, X., Usal, C., and Anegon, I.
- Published
- 2014
6. Control of Transplant Tolerance and Intragraft Regulatory T Cell Localization by Myeloid-Derived Suppressor Cells and CCL5.: Abstract# 631: Poster Board #-Session: P99-I
- Author
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Dilek, N., Poirier, N., Usal, C., Martinet, B., Blancho, G., and Vanhove, B.
- Published
- 2012
7. The Buffalo/Mna rat, an animal model of FSGS recurrence after renal transplantation
- Author
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Le Berre, L., Godfrin, Y., Perretto, S., Smit, H., Buzelin, F., Kerjaschki, D., Usal, C., Cuturi, C., Soulillou, J.P., and Dantal, J.
- Published
- 2001
- Full Text
- View/download PDF
8. Quiz case of the month
- Author
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Usal, C., Uysal, K., Kovanlıkaya, A., Çakmakçı, H., and Hazan, E.
- Published
- 2001
- Full Text
- View/download PDF
9. Tranilast, an Analogue of a Tryptophan Catabolite, Induces Allograft Tolerance By Myeloid-Derived Suppressor Cells (MDSCs).
- Author
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Menoret, S., primary, Bezie, S., additional, Li, X., additional, Usal, C., additional, and Anegon, I., additional
- Published
- 2014
- Full Text
- View/download PDF
10. Generation and Characterization of IgM, Rag1 and/or ILR2g knockout Immunodeficient Rats.
- Author
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Menoret, S., primary, Usal, C., additional, Tesson, L., additional, Remy, S., additional, Thinard, R., additional, de Cian, A., additional, Ouisse, L., additional, Fraichard, A., additional, Buelow, R., additional, Concordet, J., additional, Giovannangeli, C., additional, and Anegon, I., additional
- Published
- 2014
- Full Text
- View/download PDF
11. Tranilast, an Analogue of Tryptophan Catabolites, Induces Allograft Tolerance by CD161+ Cells
- Author
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Ménoret, S., primary, Bézie, S., additional, Li, X.-L., additional, Usal, C., additional, Caron, L., additional, and Anegon, I., additional
- Published
- 2012
- Full Text
- View/download PDF
12. Development of initial idiopathic nephrotic syndrome and post-transplantation recurrence: evidence of the same biological entity
- Author
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Le Berre, L., primary, Bruneau, S., additional, Renaudin, K., additional, Naulet, J., additional, Usal, C., additional, Smit, H., additional, Soulillou, J.-P., additional, and Dantal, J., additional
- Published
- 2010
- Full Text
- View/download PDF
13. INVOLVEMENT OF PDCS IN THE TOLEROGENIC FUNCTION OF CD8+CD45RCLOW TREG AFTER CD40-CD40L BLOCKADE IN A RAT HEART ALLOTRANSPLANTATION MODEL
- Author
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LI, X L., primary, Ménoret, S, additional, Chabannes, D, additional, Hill, M, additional, Heslan, M, additional, Usal, C, additional, Angin, M, additional, Josien, R, additional, Le Mauff, B, additional, and Anegon, I, additional
- Published
- 2008
- Full Text
- View/download PDF
14. A possible animal model for relapsing primary focal segmental glomerulosclerosis (FSGS): the Buffalo/Mna rats.
- Author
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Godfrin, Y., primary, Perretto, S., additional, Smit, H., additional, Buzelin, F., additional, Kerjaschki, D., additional, Usal, C., additional, LeBerre, L., additional, Cuturi, M. C., additional, Le Carrer, D., additional, Soulillou, J. P., additional, and Dantal, J., additional
- Published
- 1999
- Full Text
- View/download PDF
15. Transient neonatal hypoglycemia: cranial US and MRI findings.
- Author
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Cakmakci, Handan, Usal, Can, Karabay, Nuri, Kovanlikaya, Arzu, Cakmakci, H, Usal, C, Karabay, N, and Kovanlikaya, A
- Subjects
ENDOCRINE diseases ,HYPOGLYCEMIC agents ,INSULIN ,MEDICAL imaging systems ,BLOOD sugar ,GLIBENCLAMIDE ,DIAGNOSIS of brain damage ,BIRTH size ,CEREBRAL hemorrhage ,CEREBRAL ventricles ,CEREBRAL cortex ,HYPOGLYCEMIA ,MAGNETIC resonance imaging ,PROGNOSIS ,ULTRASONIC encephalography - Abstract
A case of transient neonatal hypoglycemia with patchy hyperechogenic white matter abnormalities in the frontal and parietooccipital lobes on cranial US is presented. An MRI examination revealed T1 and T2 shortening of the lesions in the occipital and frontal white matter. Follow-up cranial US demonstrated recovery of white matter changes in the patient with normal neurological outcome. [ABSTRACT FROM AUTHOR]
- Published
- 2001
- Full Text
- View/download PDF
16. Mammographic and sonographic findings in the diagnosis of idiopathic granulomatous mastitis.
- Author
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Yilmaz, Erkan, Lebe, Banu, Usal, Can, Balci, Pinar, Yilmaz, E, Lebe, B, Usal, C, and Balci, P
- Subjects
BREAST cancer ,CANCER patients ,MASTITIS ,MAMMOGRAMS ,BREAST exams ,ULTRASONIC imaging - Abstract
The aim of this study was to describe the mammographic and sonographic findings of idiopathic granulomatous mastitis which is a rare inflammatory disease of the breast of unknown etiology. The clinical, radiologic, and pathologic features of 12 cases with idiopathic granulomatous mastitis were retrospectively reviewed. Mammography was performed in all cases, 8 of which showed a focal asymmetric density, 3 had a mass with irregular margins, and 1 had no abnormality. Sonography was performed in 10 cases, and a focal area with inhomogeneous and hypoechoic pattern was depicted in 6 cases, 4 of which were associated with internal tubular hypoechoic structures. One case revealed a hypoechoic mass consistent with malignancy. In 1 case sonography showed an edematous pattern involving nearly the entire breast. Two patients had normal sonograms. If a focal asymmetric density is seen in mammography and inhomogeneous hypoechogenity with internal hypoechoic tubular structures accompany ultrasonographically, these findings should suggest the possibility of idiopathic granulomatous mastitis; however, very often idiopathic granulomatous mastitis mimics a breast carcinoma clinically and the final diagnosis should be reached histopathologically due to high false-positive and false-negative mammographic appearances. [ABSTRACT FROM AUTHOR]
- Published
- 2001
- Full Text
- View/download PDF
17. Indirect CD4+ TH1 Response, Antidonor Antibodies and Diffuse C4d Graft Deposits in Long-Term Recipients Conditioned by Donor Antigens Priming
- Author
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Ballet, C., Renaudin, K., Degauque, N., Mai, H. L., Boëffard, F., Lair, D., Berthelot, L., Feng, C., Smit, H., Usal, C., Heslan, M., Josien, R., Brouard, S., and Soulillou, J.-P.
- Abstract
Priming of recipients by DST induces long-term survival of mismatched allografts in adult rats. Despite these recipients developing inducible T regulatory cells able to transfer long-term graft survival to a secondary host, a state of chronic rejection is also observed. We revisited the molecular donor MHC targets of the cellular response in acute rejection and analyzed the cellular and humoral responses in recipients with long-term graft survival following transplantation. We found three immunodominant peptides, all derived from LEW.1W RT1.Dumolecules to be involved in acute rejection of grafts from unmodified LEW.1A recipients. Although the direct pathway of allorecognition was reduced in DST-treated recipients, the early CD4indirect pathway response to dominant peptides was almost unimpaired. We also detected early and sustained antidonor class I and II antibody subtypes with diffuse C4d deposits on graft vessels. Finally, long-term accepted grafts displayed leukocyte infiltration, endarteritis and fibrosis, which evolved toward vascular narrowing at day 100. Altogether, these data suggest that the chronic graft lesions developed in long-term graft recipients are the result of progressive humoral injury associated with a persisting indirect T helper response. These features may represent a useful model for understanding and manipulating chronic active antibody-mediated rejection in human.
- Published
- 2009
- Full Text
- View/download PDF
18. Indirect CD4+TH1 Response, Antidonor Antibodies and Diffuse C4d Graft Deposits in Long-Term Recipients Conditioned by Donor Antigens Priming
- Author
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Ballet, C., Renaudin, K., Degauque, N., Mai, H.L., Boëffard, F., Lair, D., Berthelot, L., Feng, C., Smit, H., Usal, C., Heslan, M., Josien, R., Brouard, S., and Soulillou, J.-P.
- Abstract
Priming of recipients by DST induces long-term survival of mismatched allografts in adult rats. Despite these recipients developing inducible T regulatory cells able to transfer long-term graft survival to a secondary host, a state of chronic rejection is also observed. We revisited the molecular donor MHC targets of the cellular response in acute rejection and analyzed the cellular and humoral responses in recipients with long-term graft survival following transplantation. We found three immunodominant peptides, all derived from LEW.1W RT1.Dumolecules to be involved in acute rejection of grafts from unmodified LEW.1A recipients. Although the direct pathway of allorecognition was reduced in DST-treated recipients, the early CD4+indirect pathway response to dominant peptides was almost unimpaired. We also detected early and sustained antidonor class I and II antibody subtypes with diffuse C4d deposits on graft vessels. Finally, long-term accepted grafts displayed leukocyte infiltration, endarteritis and fibrosis, which evolved toward vascular narrowing at day 100. Altogether, these data suggest that the chronic graft lesions developed in long-term graft recipients are the result of progressive humoral injury associated with a persisting indirect T helper response. These features may represent a useful model for understanding and manipulating chronic active antibody-mediated rejection in human.
- Published
- 2009
- Full Text
- View/download PDF
19. Induction of Donor-Specific Allograft Tolerance by Short-Term Treatment with LF15-0195 After Transplantation. Evidence for a Direct Effect on T-Cell Differentiation
- Author
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Chiffoleau, E., Bériou, G., Dutartre, P., Usal, C., Soulillou, J-P., and Cuturi, M.C.
- Abstract
A 20-day treatment with LF15-0195, a deoxyspergualine analog, induced long-term heart allograft survival in the rat without signs of chronic rejection. LF15-0195-treated recipients did not develop an anti-donor alloantibody response. Analysis of graft-infiltrating cells, IL10, TNFα, IFNγ mRNA and iNOS protein expression in allografts, 5 days after transplantation, showed that they were markedly decreased in allografts from LF15-0195-treated recipients compared with allografts from untreated recipients. Surprisingly, spleen T cells from LF15-0195 recipients, 5 days after grafting, were able to proliferate strongly in vitro, when stimulated with donor cells, but had reduced mRNA expression for IFNγ compared with spleen T cells from untreated graft recipients. Furthermore, when T cells from naive animals were stimulated in vitro, using anti-CD3 and anti-CD28, LF15-0195 also increased T-cell proliferation in a dose-dependent fashion; however, these cells expressed less of the Th1-related cytokines, IFNγ and IL2, compared with untreated cells, suggesting that LF15-0195 could act on T-cell differentiation. In conclusion, we show here that a short-term treatment with LF15-0195 induced long-term allograft tolerance, decreasing the in situ anti-donor response, and we illustrate evidence for the development of regulatory mechanisms.
- Published
- 2002
- Full Text
- View/download PDF
20. Sonographic and MRI findings in prepubertal adnexal hemorrhagic cyst with torsion.
- Author
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Yilmaz, Erkan, Usal, Can, Kovanlikaya, Arzu, Karabay, Nuri, Yilmaz, E, Usal, C, Kovanlikaya, A, and Karabay, N
- Published
- 2001
- Full Text
- View/download PDF
21. The Buffalo/Mna rat, an animal model of FSGS recurrence after renal transplantation
- Author
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Berre, L. Le, Godfrin, Y., Perretto, S., Smit, H., Buzelin, F., Kerjaschki, D., Usal, C., Cuturi, C., Soulillou, J. P., and Dantal, J.
- Published
- 2001
- Full Text
- View/download PDF
22. Control of transplant tolerance and intragraft regulatory T cell localization by CCL5
- Author
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Dilek Nahzli, Poirier Nicolas, Usal Claire, Martinet Bernard, Blancho Gilles, and Vanhove Bernard
- Subjects
Medicine - Published
- 2012
- Full Text
- View/download PDF
23. Immunosuppressive role of fibrinogen-like protein 2 (FGL2) in CD8+regulatory T cells-mediated long-term graft survival
- Author
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Bézie Séverine, Ménoret Séverine, Tesson Laurent, Li Xian-Liang, Usal Claire, Anegon Ignacio, and Caron Lise
- Subjects
Medicine - Published
- 2011
- Full Text
- View/download PDF
24. Tranilast, an Analogue of Tryptophan Catabolites, Induces Allograft Tolerance by CD161+ Cells.
- Author
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Ménoret, S., Bézie, S., Li, X.-L., Usal, C., Caron, L., and Anegon, I.
- Published
- 2012
- Full Text
- View/download PDF
25. Functional connexion of bacterioferritin in antibiotic production and morphological differentiation in Streptomyces coelicolor.
- Author
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García-Martín J, García-Abad L, Santamaría RI, and Díaz M
- Subjects
- Cytochrome b Group metabolism, Cytochrome b Group genetics, Gene Expression Regulation, Bacterial, Prodigiosin metabolism, Prodigiosin analogs & derivatives, Prodigiosin biosynthesis, Reactive Oxygen Species metabolism, Proteomics, Benzoisochromanequinones, Streptomyces coelicolor metabolism, Streptomyces coelicolor genetics, Streptomyces coelicolor growth & development, Anti-Bacterial Agents biosynthesis, Anti-Bacterial Agents metabolism, Ferritins metabolism, Ferritins genetics, Bacterial Proteins metabolism, Bacterial Proteins genetics, Iron metabolism, Anthraquinones metabolism
- Abstract
Background: Several two-component systems of Streptomyces coelicolor, a model organism used for studying antibiotic production in Streptomyces, affect the expression of the bfr (SCO2113) gene that encodes a bacterioferritin, a protein involved in iron storage. In this work, we have studied the effect of the deletion mutant ∆bfr in S. coelicolor., Results: The ∆bfr mutant exhibits a delay in morphological differentiation and produces a lesser amount of the two pigmented antibiotics (actinorhodin and undecylprodigiosin) compared to the wild type on complex media. The effect of iron in minimal medium was tested in the wild type and ∆bfr mutant. Consequently, we also observed different levels of production of the two pigmented antibiotics between the two strains, depending on the iron concentration and the medium (solid or liquid) used. Contrary to expectations, no differences in intracellular iron concentration were detected between the wild type and ∆bfr mutant. However, a higher level of reactive oxygen species in the ∆bfr mutant and a higher tolerance to oxidative stress were observed. Proteomic analysis showed no variation in iron response proteins, but there was a lower abundance of proteins related to actinorhodin and ribosomal proteins, as well as others related to secondary metabolite production and differentiation. Additionally, a higher abundance of proteins related to various types of stress, such as respiration and hypoxia among others, was also revealed. Data are available via ProteomeXchange with identifier PXD050869., Conclusion: This bacterioferritin in S. coelicolor (Bfr) is a new element in the complex regulation of secondary metabolism in S. coelicolor and, additionally, iron acts as a signal to modulate the biosynthesis of active molecules. Our model proposes an interaction between Bfr and iron-containing regulatory proteins. Thus, identifying these interactions would provide new information for improving antibiotic production in Streptomyces., (© 2024. The Author(s).)
- Published
- 2024
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26. CD4 + and CD8 + regulatory T cell characterization in the rat using a unique transgenic Foxp3-EGFP model.
- Author
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Ménoret S, Tesson L, Remy S, Gourain V, Sérazin C, Usal C, Guiffes A, Chenouard V, Ouisse LH, Gantier M, Heslan JM, Fourgeux C, Poschmann J, Guillonneau C, and Anegon I
- Subjects
- Rats, Animals, Interleukin-2 genetics, Interleukin-2 metabolism, Transforming Growth Factor beta metabolism, Forkhead Transcription Factors genetics, Forkhead Transcription Factors metabolism, T-Lymphocytes, Regulatory metabolism, CD8-Positive T-Lymphocytes metabolism
- Abstract
Background: Regulatory T cells (Treg) in diverse species include CD4
+ and CD8+ T cells. In all species, CD8+ Treg have been only partially characterized and there is no rat model in which CD4+ and CD8+ FOXP3+ Treg are genetically tagged., Results: We generated a Foxp3-EGFP rat transgenic line in which FOXP3 gene was expressed and controlled EGFP. CD4+ and CD8+ T cells were the only cells that expressed EGFP, in similar proportion as observed with anti-FOXP3 antibodies and co-labeled in the same cells. CD4+ EGFP+ Treg were 5-10 times more frequent than CD8+ EGFP+ Treg. The suppressive activity of CD4+ and CD8+ Treg was largely confined to EGFP+ cells. RNAseq analyses showed similarities but also differences among CD4+ and CD8+ EGFP+ cells and provided the first description of the natural FOXP3+ CD8+ Treg transcriptome. In vitro culture of CD4+ and CD8+ EGFP- cells with TGFbeta and IL-2 generated induced EGFP+ Treg. CD4+ and CD8+ EGFP+ Treg were expanded upon in vivo administration of a low dose of IL-2., Conclusions: This new and unique rat line constitutes a useful model to identify and isolate viable CD4+ and CD8+ FOXP3+ Treg. Additionally, it allows to identify molecules expressed in CD8+ Treg that may allow to better define their phenotype and function not only in rats but also in other species., (© 2023. The Author(s).)- Published
- 2023
- Full Text
- View/download PDF
27. The Role of S. cerevisiae Sub1/PC4 in Transcription Elongation Depends on the C-Terminal Region and Is Independent of the ssDNA Binding Domain.
- Author
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Collin A, González-Jiménez A, González-Jiménez MDC, Alfonso MJ, and Calvo O
- Subjects
- Humans, DNA-Binding Proteins metabolism, RNA Polymerase II metabolism, Transcription Factors genetics, Transcription Factors metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Saccharomyces cerevisiae Sub1 (ScSub1) has been defined as a transcriptional stimulatory protein due to its homology to the ssDNA binding domain (ssDBD) of human PC4 (hPC4). Recently, PC4/Sub1 orthologues have been elucidated in eukaryotes, prokaryotes, and bacteriophages with functions related to DNA metabolism. Additionally, ScSub1 contains a unique carboxyl-terminal region (CT) of unknown function up to date. Specifically, it has been shown that Sub1 is required for transcription activation, as well as other processes, throughout the transcription cycle. Despite the progress that has been made in understanding the mechanism underlying Sub1's functions, some questions remain unanswered. As a case in point: whether Sub1's roles in initiation and elongation are differentially predicated on distinct regions of the protein or how Sub1's functions are regulated. Here, we uncover some residues that are key for DNA-ScSub1 interaction in vivo, localized in the ssDBD, and required for Sub1 recruitment to promoters. Furthermore, using an array of genetic and molecular techniques, we demonstrate that the CT region is required for transcription elongation by RNA polymerase II (RNAPII). Altogether, our data indicate that Sub1 plays a dual role during transcription-in initiation through the ssDBD and in elongation through the CT region.
- Published
- 2022
- Full Text
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28. IL-34 deficiency impairs FOXP3 + Treg function in a model of autoimmune colitis and decreases immune tolerance homeostasis.
- Author
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Freuchet A, Salama A, Bézie S, Tesson L, Rémy S, Humeau R, Règue H, Sérazin C, Flippe L, Peterson P, Vimond N, Usal C, Ménoret S, Heslan JM, Duteille F, Blanchard F, Giral M, Colonna M, Anegon I, and Guillonneau C
- Subjects
- Animals, Forkhead Transcription Factors, Homeostasis, Humans, Immune Tolerance, Mice, Rats, Colitis immunology, Graft vs Host Disease immunology, Interleukins deficiency, Interleukins genetics, T-Lymphocytes, Regulatory immunology
- Abstract
Background: Immune homeostasis requires fully functional Tregs with a stable phenotype to control autoimmunity. Although IL-34 is a cytokine first described as mainly involved in monocyte cell survival and differentiation, we recently described its expression by CD8
+ Tregs in a rat model of transplantation tolerance and by activated FOXP3+ CD4+ and CD8+ Tregs in human healthy individuals. However, its role in autoimmunity and potential in human diseases remains to be determined., Methods: We generated Il34-/- rats and using both Il34-/- rats and mice, we investigated their phenotype under inflammatory conditions. Using Il34-/- rats, we further analyzed the impact of the absence of expression of IL-34 for CD4+ Tregs suppressive function. We investigated the potential of IL-34 in human disease to prevent xenogeneic GVHD and human skin allograft rejection in immune humanized immunodeficient NSG mice. Finally, taking advantage of a biocollection, we investigated the correlation between presence of IL-34 in the serum and kidney transplant rejection., Results: Here we report that the absence of expression of IL-34 in Il34-/- rats and mice leads to an unstable immune phenotype, with production of multiple auto-antibodies, exacerbated under inflammatory conditions with increased susceptibility to DSS- and TNBS-colitis in Il34-/- animals. Moreover, we revealed the striking inability of Il34-/- CD4+ Tregs to protect Il2rg-/- rats from a wasting disease induced by transfer of pathogenic cells, in contrast to Il34+/+ CD4+ Tregs. We also showed that IL-34 treatment delayed EAE in mice as well as GVHD and human skin allograft rejection in immune humanized immunodeficient NSG mice. Finally, we show that presence of IL-34 in the serum is associated with a longer rejection-free period in kidney transplanted patients., Conclusion: Altogether, our data emphasize on the crucial necessity of IL-34 for immune homeostasis and for CD4+ Tregs suppressive function. Our data also shows the therapeutic potential of IL-34 in human transplantation and auto-immunity., Highlights: -Absence of expression of IL-34 in Il34-/- rats and mice leads to an unstable immune phenotype, with a production of multiple auto-antibodies and exacerbated immune pathology under inflammatory conditions. -Il34-/- CD4+ Tregs are unable to protect Il2rg-/- rats from colitis induced by transfer of pathogenic cells. -IL-34 treatment delayed EAE in mice, as well as acute GVHD and human skin allograft rejection in immune-humanized immunodeficient NSG mice., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)- Published
- 2022
- Full Text
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29. Angular-Resolved Thomson Parabola Spectrometer for Laser-Driven Ion Accelerators.
- Author
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Salgado-López C, Apiñaniz JI, Henares JL, Pérez-Hernández JA, de Luis D, Volpe L, and Gatti G
- Abstract
This article reports the development, construction, and experimental test of an angle-resolved Thomson parabola (TP) spectrometer for laser-accelerated multi-MeV ion beams in order to distinguish between ionic species with different charge-to-mass ratio. High repetition rate (HHR) compatibility is guaranteed by the use of a microchannel plate (MCP) as active particle detector. The angular resolving power, which is achieved due to an array of entrance pinholes, can be simply adjusted by modifying the geometry of the experiment and/or the pinhole array itself. The analysis procedure allows for different ion traces to cross on the detector plane, which greatly enhances the flexibility and capabilities of the detector. A full characterization of the TP magnetic field is implemented into a relativistic code developed for the trajectory calculation of each pinhole beamlet. We describe the first test of the spectrometer at the 1PW VEGA 3 laser facility at CLPU, Salamanca (Spain), where up to 15MeV protons and carbon ions from a 3μm laser-irradiated Al foil are detected.
- Published
- 2022
- Full Text
- View/download PDF
30. Anti-CD45RC antibody immunotherapy prevents and treats experimental autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome.
- Author
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Besnard M, Sérazin C, Ossart J, Moreau A, Vimond N, Flippe L, Sein H, Smith GA, Pittaluga S, Ferré EM, Usal C, Anegon I, Ranki A, Lionakis MS, Peterson P, and Guillonneau C
- Subjects
- Animals, Autoantibodies, Humans, Immunotherapy, Rats, T-Lymphocytes, Regulatory, Autoimmune Diseases, Polyendocrinopathies, Autoimmune genetics, Polyendocrinopathies, Autoimmune therapy
- Abstract
Targeted monoclonal antibody (mAb) therapies show great promise for the treatment of transplant rejection and autoimmune diseases by inducing more specific immunomodulatory effects than broadly immunosuppressive drugs routinely used. We recently described the therapeutic advantage of targeting CD45RC, expressed at high levels by conventional T (Tconv) cells (CD45RChi), their precursors, and terminally differentiated T (TEMRA) cells, but not by regulatory T cells (Tregs; CD45RClo/-). We demonstrated efficacy of anti-CD45RC mAb treatment in transplantation, but its potential has not been examined in autoimmune diseases. Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare genetic syndrome caused by loss-of-function mutations of autoimmune regulator (AIRE), a key central tolerance mediator, leading to abnormal autoreactive T cell responses and autoantibody production. Herein, we show that, in a rat model of APECED syndrome, anti-CD45RC mAb was effective for both prevention and treatment of autoimmune manifestations and inhibited autoantibody development. Anti-CD45RC mAb intervention depleted CD45RChi T cells, inhibited CD45RChi B cells, and restored the Treg/Tconv cell ratio and the altered Treg transcriptomic profile. In APECED patients, CD45RC was significantly increased in peripheral blood T cells, and lesioned organs from APECED patients were infiltrated by CD45RChi cells. Our observations highlight the potential role for CD45RChi cells in the pathogenesis of experimental and human APECED syndrome and the potential of anti-CD45RC antibody treatment.
- Published
- 2022
- Full Text
- View/download PDF
31. Genome-wide sequencing analysis of Sgs1, Exo1, Rad51, and Srs2 in DNA repair by homologous recombination.
- Author
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Ramos F, Durán L, Sánchez M, Campos A, Hernández-Villamor D, Antequera F, and Clemente-Blanco A
- Subjects
- DNA Breaks, Double-Stranded, DNA Damage, DNA Helicases genetics, DNA Helicases metabolism, DNA Repair genetics, DNA Repair physiology, DNA Replication, Exodeoxyribonucleases genetics, Genome-Wide Association Study, Genomic Instability, Rad51 Recombinase genetics, RecQ Helicases genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Sequence Analysis, DNA methods, Recombinational DNA Repair genetics, Recombinational DNA Repair physiology
- Abstract
Homologous recombination is essential to maintain genome stability in response to DNA damage. Here, we have used genome-wide sequencing to quantitatively analyze at nucleotide resolution the dynamics of DNA end resection, re-synthesis, and gene conversion at a double-strand break. Resection initiates asymmetrically in an MRX-independent manner before proceeding steadily in both directions. Sgs1, Exo1, Rad51, and Srs2 differently regulate the rate and symmetry of early and late resection. Exo1 also ensures the coexistence of resection and re-synthesis, while Srs2 guarantees a constant and symmetrical DNA re-polymerization. Gene conversion is MMR independent, spans only a minor fraction of the resected region, and its unidirectionality depends on Srs2. Finally, these repair factors prevent the development of alterations remote from the DNA lesion, such as subtelomeric instability, duplication of genomic regions, and over-replication of Ty elements. Altogether, this approach allows a quantitative analysis and a direct genome-wide visualization of DNA repair by homologous recombination., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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- View/download PDF
32. RNA polymerase II phosphorylation and gene looping: new roles for the Rpb4/7 heterodimer in regulating gene expression.
- Author
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Calvo O
- Subjects
- Phosphorylation, RNA Polymerase II genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Gene Expression Regulation, Fungal, RNA Polymerase II metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Transcription, Genetic
- Abstract
In eukaryotes, cellular RNAs are produced by three nuclear RNA polymerases (RNAPI, II, and III), which are multisubunit complexes. They share structural and functional features, although they are specialized in the synthesis of specific RNAs. RNAPII transcribes the vast majority of cellular RNAs, including mRNAs and a large number of noncoding RNAs. The structure of RNAPII is highly conserved in all eukaryotes, consisting of 12 subunits (Rpb1-12) organized into five structural modules, among which the Rpb4 and Rpb7 subunits form the stalk. Early studies suggested an accessory role for Rpb4, because is required for specific gene transcription pathways. Far from this initial hypothesis, it is now well established that the Rpb4/7 heterodimer plays much wider roles in gene expression regulation. It participates in nuclear and cytosolic processes ranging from transcription to translation and mRNA degradation in a cyclical process. For this reason, Rpb4/7 is considered a coordinator of gene expression. New functions have been added to the list of stalk functions during transcription, which will be reviewed herein: first, a role in the maintenance of proper RNAPII phosphorylation levels, and second, a role in the establishment of a looped gene architecture in actively transcribed genes.
- Published
- 2020
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33. In Vivo Analysis of Human Immune Responses in Immunodeficient Rats.
- Author
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Ménoret S, Ouisse LH, Tesson L, Remy S, Usal C, Guiffes A, Chenouard V, Royer PJ, Evanno G, Vanhove B, Piaggio E, and Anegon I
- Subjects
- Animals, Antigens, Differentiation genetics, Antilymphocyte Serum pharmacology, Breast Neoplasms immunology, Breast Neoplasms pathology, Breast Neoplasms therapy, Cell Line, Tumor, Disease Models, Animal, Female, Graft vs Host Disease drug therapy, Graft vs Host Disease genetics, Graft vs Host Disease immunology, Heterografts, Homeodomain Proteins genetics, Humans, Immunoglobulin gamma-Chains genetics, Immunologic Deficiency Syndromes genetics, Leukocytes, Mononuclear immunology, Rats, Sprague-Dawley, Rats, Transgenic, Receptors, Immunologic genetics, Xenograft Model Antitumor Assays, Antigens, Differentiation immunology, Homeodomain Proteins immunology, Immunocompromised Host, Immunoglobulin gamma-Chains immunology, Immunologic Deficiency Syndromes immunology, Leukocytes, Mononuclear transplantation, Receptors, Immunologic immunology
- Abstract
Background: Humanized immune system immunodeficient mice have been extremely useful for the in vivo analyses of immune responses in a variety of models, including organ transplantation and graft versus host disease (GVHD) but they have limitations. Rat models are interesting complementary alternatives presenting advantages over mice, such as their size and their active complement compartment. Immunodeficient rats have been generated but human immune responses have not yet been described., Methods: We generated immunodeficient Rat Rag-/- Gamma chain-/- human signal regulatory protein alpha-positive (RRGS) rats combining Rag1 and Il2rg deficiency with the expression of human signal regulatory protein alpha, a negative regulator of macrophage phagocytosis allowing repression of rat macrophages by human CD47-positive cells. We then immune humanized RRGS animals with human peripheral blood mononuclear cells (hPBMCs) to set up a human acute GVHD model. Treatment of GVHD was done with a new porcine antihuman lymphocyte serum active through complement-dependent cytotoxicity. We also established a tumor xenograft rejection model in these hPBMCs immune system RRGS animals by subcutaneous implantation of a human tumor cell line., Results: RRGS animals receiving hPBMCs showed robust and reproducible reconstitution, mainly by T and B cells. A dose-dependent acute GVHD process was observed with progressive weight loss, tissue damage, and death censoring. Antihuman lymphocyte serum (L1S1) antibody completely prevented acute GVHD. In the human tumor xenograft model, detectable tumors were rejected upon hPBMCs injection., Conclusions: hPBMC can be implanted in RRGS animals and elicit acute GVHD or rejection of human tumor cells and these are useful models to test new immunotherapies.
- Published
- 2020
- Full Text
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34. Cross-Reactive Donor-Specific CD8 + Tregs Efficiently Prevent Transplant Rejection.
- Author
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Picarda E, Bézie S, Usero L, Ossart J, Besnard M, Halim H, Echasserieau K, Usal C, Rossjohn J, Bernardeau K, Gras S, and Guillonneau C
- Subjects
- Allografts immunology, Amino Acid Motifs, Amino Acid Sequence, Animals, Consensus Sequence, Histocompatibility Antigens Class I immunology, Histocompatibility Antigens Class II chemistry, Histocompatibility Antigens Class II immunology, Humans, Leukocyte Common Antigens metabolism, Lymphocyte Activation immunology, Peptides chemistry, Peptides immunology, Rats, Vaccination, CD8-Positive T-Lymphocytes immunology, Cross Reactions immunology, Graft Rejection immunology, T-Lymphocytes, Regulatory immunology, Tissue Donors
- Abstract
To reduce the use of non-specific immunosuppressive drugs detrimental to transplant patient health, therapies in development aim to achieve antigen-specific tolerance by promoting antigen-specific regulatory T cells (Tregs). However, identification of the natural antigens recognized by Tregs and the contribution of their dominance in transplantation has been challenging. We identify epitopes derived from distinct major histocompatibility complex (MHC) class II molecules, sharing a 7-amino acid consensus sequence positioned in a central mobile section in complex with MHC class I, recognized by cross-reactive CD8
+ Tregs, enriched in the graft. Antigen-specific CD8+ Tregs can be induced in vivo with a 16-amino acid-long peptide to trigger transplant tolerance. Peptides derived from human HLA class II molecules, harboring the rat consensus sequence, also activate and expand human CD8+ Tregs, suggesting its potential in human transplantation. Altogether, this work should facilitate the development of therapies with peptide epitopes for transplantation and improve our understanding of CD8+ Treg recognition., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2019
- Full Text
- View/download PDF
35. SIRPα/CD47 axis controls the maintenance of transplant tolerance sustained by myeloid-derived suppressor cells.
- Author
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Pengam S, Durand J, Usal C, Gauttier V, Dilek N, Martinet B, Daguin V, Mary C, Thepenier V, Teppaz G, Renaudin K, Blancho G, Vanhove B, and Poirier N
- Subjects
- Animals, Antibodies, Monoclonal administration & dosage, CD47 Antigen antagonists & inhibitors, CD47 Antigen immunology, Chemokines, Graft Rejection pathology, Graft Survival immunology, Myeloid Cells cytology, Rats, Receptors, Immunologic antagonists & inhibitors, Receptors, Immunologic immunology, CD47 Antigen metabolism, Graft Rejection immunology, Kidney Transplantation adverse effects, Myeloid Cells immunology, Myeloid-Derived Suppressor Cells immunology, Receptors, Immunologic metabolism, Transplantation Tolerance immunology
- Abstract
Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature hematopoietic precursors known to suppress immune responses. Interaction of SIRP alpha (SIRPα), expressed by myeloid cells, with the ubiquitous receptor CD47 is an important immune checkpoint of the innate response regulating macrophages and dendritic cells functions. We previously described that MDSC expressing SIRPα accumulated after transplantation and maintained kidney allograft tolerance. However, the role of the SIRPα/CD47 axis on MDSC function remained unknown. Here, we found that blocking SIRPα or CD47 with monoclonal antibodies (mAbs) induced differentiation of MDSC into myeloid cells overexpressing MHC class II, CD86 costimulatory molecule and increased secretion of macrophage-recruiting chemokines (eg, MCP-1). Using a model of long-term kidney allograft tolerance sustained by MDSC, we observed that administration of blocking anti-SIRPα or CD47 mAbs induced graft dysfunction and rejection. Loss of tolerance came along with significant decrease of MDSC and increase in MCP-1 concentration in the periphery. Graft histological and transcriptomic analyses revealed an inflammatory (M1) macrophagic signature at rejection associated with overexpression of MCP-1 mRNA and protein in the graft. These findings indicate that the SIRPα-CD47 axis regulates the immature phenotype and chemokine secretion of MDSC and contributes to the induction and the active maintenance of peripheral acquired immune tolerance., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)
- Published
- 2019
- Full Text
- View/download PDF
36. Characterization of two rat models of cystic fibrosis-KO and F508del CFTR-Generated by Crispr-Cas9.
- Author
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Dreano E, Bacchetta M, Simonin J, Galmiche L, Usal C, Slimani L, Sadoine J, Tesson L, Anegon I, Concordet JP, Hatton A, Vignaud L, Tondelier D, Sermet-Gaudelus I, Chanson M, and Cottart CH
- Abstract
Background: Genetically engineered animals are essential for gaining a proper understanding of the disease mechanisms of cystic fibrosis (CF). The rat is a relevant laboratory model for CF because of its zootechnical capacity, size, and airway characteristics, including the presence of submucosal glands., Methods: We describe the generation of a CF rat model (F508del) homozygous for the p.Phe508del mutation in the transmembrane conductance regulator ( Cftr ) gene. This model was compared to new Cftr
-/- rats (CFTR KO). Target organs in CF were examined by histological staining of tissue sections and tooth enamel was quantified by micro-computed tomography. The activity of CFTR was evaluated by nasal potential difference (NPD) and short-circuit current measurements. The effect of VX-809 and VX-770 was analyzed on nasal epithelial primary cell cultures from F508del rats., Results: Both newborn F508del and Knock out (KO) animals developed intestinal obstruction that could be partly compensated by special diet combined with an osmotic laxative. The two rat models exhibited CF phenotypic anomalies such as vas deferens agenesis and tooth enamel defects. Histology of the intestine, pancreas, liver, and lungs was normal. Absence of CFTR function in KO rats was confirmed ex vivo by short-circuit current measurements on colon mucosae and in vivo by NPD, whereas residual CFTR activity was observed in F508del rats. Exposure of F508del CFTR nasal primary cultures to a combination of VX-809 and VX-770 improved CFTR-mediated Cl- transport., Conclusions: The F508del rats reproduce the phenotypes observed in CFTR KO animals and represent a novel resource to advance the development of CF therapeutics., Competing Interests: Dr. Sermet‐Gaudelus reports grants from Vertex Therapeutics, personal fees from Vertex Therapeutics, personal fees from Eloxx, nonfinancial support from PTC Therapeutics, outside the submitted work., (© 2019 The Authors. Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.)- Published
- 2019
- Full Text
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37. PP4 phosphatase cooperates in recombinational DNA repair by enhancing double-strand break end resection.
- Author
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Villoria MT, Gutiérrez-Escribano P, Alonso-Rodríguez E, Ramos F, Merino E, Campos A, Montoya A, Kramer H, Aragón L, and Clemente-Blanco A
- Subjects
- DNA Replication, DNA, Fungal metabolism, Phosphorylation, Phosphoserine metabolism, DNA Breaks, Double-Stranded, Phosphoprotein Phosphatases metabolism, Recombinational DNA Repair, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The role of Rad53 in response to a DNA lesion is central for the accurate orchestration of the DNA damage response. Rad53 activation relies on its phosphorylation by Mec1 and its own autophosphorylation in a manner dependent on the adaptor Rad9. While the mechanism behind Rad53 activation has been well documented, less is known about the processes that counteract its activity along the repair of a DNA adduct. Here, we describe that PP4 phosphatase is required to avoid Rad53 hyper-phosphorylation during the repair of a double-strand break, a process that impacts on the phosphorylation status of multiple factors involved in the DNA damage response. PP4-dependent Rad53 dephosphorylation stimulates DNA end resection by relieving the negative effect that Rad9 exerts over the Sgs1/Dna2 exonuclease complex. Consequently, elimination of PP4 activity affects resection and repair by single-strand annealing, defects that are bypassed by reducing Rad53 hyperphosphorylation. These results confirm that Rad53 phosphorylation is controlled by PP4 during the repair of a DNA lesion and demonstrate that the attenuation of its kinase activity during the initial steps of the repair process is essential to efficiently enhance recombinational DNA repair pathways that depend on long-range resection for their success., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2019
- Full Text
- View/download PDF
38. Role of protein phosphatases PP1, PP2A, PP4 and Cdc14 in the DNA damage response.
- Author
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Ramos F, Villoria MT, Alonso-Rodríguez E, and Clemente-Blanco A
- Abstract
Maintenance of genome integrity is fundamental for cellular physiology. Our hereditary information encoded in the DNA is intrinsically susceptible to suffer variations, mostly due to the constant presence of endogenous and environmental genotoxic stresses. Genomic insults must be repaired to avoid loss or inappropriate transmission of the genetic information, a situation that could lead to the appearance of developmental anomalies and tumorigenesis. To safeguard our genome, cells have evolved a series of mechanisms collectively known as the DNA damage response (DDR). This surveillance system regulates multiple features of the cellular response, including the detection of the lesion, a transient cell cycle arrest and the restoration of the broken DNA molecule. While the role of multiple kinases in the DDR has been well documented over the last years, the intricate roles of protein dephosphorylation have only recently begun to be addressed. In this review, we have compiled recent information about the function of protein phosphatases PP1, PP2A, PP4 and Cdc14 in the DDR, focusing mainly on their capacity to regulate the DNA damage checkpoint and the repair mechanism encompassed in the restoration of a DNA lesion., Competing Interests: Conflict of interest: The authors declare no competing financial interests. Correspondence should be addressed to A.C-B.
- Published
- 2019
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39. Generation of Immunodeficient Rats With Rag1 and Il2rg Gene Deletions and Human Tissue Grafting Models.
- Author
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Ménoret S, Ouisse LH, Tesson L, Delbos F, Garnier D, Remy S, Usal C, Concordet JP, Giovannangeli C, Chenouard V, Brusselle L, Merieau E, Nerrière-Daguin V, Duteille F, Bellier-Waast F, Fraichard A, Nguyen TH, and Anegon I
- Subjects
- Animals, Animals, Genetically Modified, Crosses, Genetic, Disease Models, Animal, Exons, Female, Genotype, Hepatocytes cytology, Humans, Immune System, Liver immunology, Male, Mutation, Rats, Rats, Sprague-Dawley, Skin Transplantation, Transplantation, Heterologous, Transplants, Gene Deletion, Homeodomain Proteins genetics, Interleukin Receptor Common gamma Subunit genetics
- Abstract
Background: Immunodeficient mice are invaluable tools to analyze the long-term effects of potentially immunogenic molecules in the absence of adaptive immune responses. Nevertheless, there are models and experimental situations that would beneficiate of larger immunodeficient recipients. Rats are ideally suited to perform experiments in which larger size is needed and are still a small animal model suitable for rodent facilities. Additionally, rats reproduce certain human diseases better than mice, such as ankylosing spondylitis and Duchenne disease, and these disease models would greatly benefit from immunodeficient rats to test different immunogenic treatments., Methods: We describe the generation of Il2rg-deficient rats and their crossing with previously described Rag1-deficient rats to generate double-mutant RRG animals., Results: As compared with Rag1-deficient rats, Il2rg-deficient rats were more immunodeficient because they partially lacked not only T and B cells but also NK cells. RRG animals showed a more profound immunossuppressed phenotype because they displayed undetectable levels of T, B, and NK cells. Similarly, all immunoglobulin isotypes in sera were decreased in Rag1- or Il2rg-deficient rats and undetectable in Rats Rag1 and Il2rg (RRG) animals. Rag1- or Il2rg-deficient rats rejected allogeneic skin transplants and human tumors, whereas animals not only accepted allogeneic rat skin but also xenogeneic human tumors, skin, and hepatocytes. Immune humanization of RRG animals was unsuccessful., Conclusions: Thus, immunodeficient RRG animals are useful recipients for long-term studies in which immune responses could be an obstacle, including tissue humanization of different tissues.
- Published
- 2018
- Full Text
- View/download PDF
40. Generation of gene-edited rats by delivery of CRISPR/Cas9 protein and donor DNA into intact zygotes using electroporation.
- Author
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Remy S, Chenouard V, Tesson L, Usal C, Ménoret S, Brusselle L, Heslan JM, Nguyen TH, Bellien J, Merot J, De Cian A, Giovannangeli C, Concordet JP, and Anegon I
- Subjects
- Animals, Clustered Regularly Interspaced Short Palindromic Repeats genetics, Electroporation methods, Female, Genotype, Microscopy, Confocal, Mutation, Rats, CRISPR-Associated Protein 9 metabolism, Clustered Regularly Interspaced Short Palindromic Repeats physiology, Zygote metabolism
- Abstract
The generation of gene-edited animals using the CRISPRs/Cas9 system is based on microinjection into zygotes which is inefficient, time consuming and demands high technical skills. We report the optimization of an electroporation method for intact rat zygotes using sgRNAs and Cas9 protein in combination or not with ssODNs (~100 nt). This resulted in high frequency of knockouts, between 15 and 50% of analyzed animals. Importantly, using ssODNs as donor template resulted in precise knock-in mutations in 25-100% of analyzed animals, comparable to microinjection. Electroporation of long ssDNA or dsDNA donors successfully used in microinjection in the past did not allow generation of genome-edited animals despite dsDNA visualization within zygotes. Thus, simultaneous electroporation of a large number of intact rat zygotes is a rapid, simple, and efficient method for the generation of a variety of genome-edited rats.
- Published
- 2017
- Full Text
- View/download PDF
41. Advances in transgenic animal models and techniques.
- Author
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Ménoret S, Tesson L, Remy S, Usal C, Ouisse LH, Brusselle L, Chenouard V, and Anegon I
- Subjects
- Animals, Humans, Animals, Genetically Modified genetics, Gene Transfer Techniques trends, Models, Animal
- Abstract
On May 11th and 12th 2017 was held in Nantes, France, the international meeting "Advances in transgenic animal models and techniques" ( http://www.trm.univ-nantes.fr/ ). This biennial meeting is the fifth one of its kind to be organized by the Transgenic Rats ImmunoPhenomic (TRIP) Nantes facility ( http://www.tgr.nantes.inserm.fr/ ). The meeting was supported by private companies (SONIDEL, Scionics computer innovation, New England Biolabs, MERCK, genOway, Journal Disease Models and Mechanisms) and by public institutions (International Society for Transgenic Technology, University of Nantes, INSERM UMR 1064, SFR François Bonamy, CNRS, Région Pays de la Loire, Biogenouest, TEFOR infrastructure, ITUN, IHU-CESTI and DHU-Oncogeffe and Labex IGO). Around 100 participants, from France but also from different European countries, Japan and USA, attended the meeting.
- Published
- 2017
- Full Text
- View/download PDF
42. In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients.
- Author
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Bézie S, Usal C, and Guillonneau C
- Subjects
- Animals, Coculture Techniques, Heart Transplantation methods, Humans, Immunity, Humoral, Rats, Transplant Recipients, Transplantation, Homologous methods, Adoptive Transfer methods, B-Lymphocytes immunology, Immune Tolerance immunology, Myeloid Cells immunology, T-Lymphocytes, Regulatory immunology
- Abstract
The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.
- Published
- 2017
- Full Text
- View/download PDF
43. Cdc14 and Chromosome Condensation: Evaluation of the Recruitment of Condensin to Genomic Regions.
- Author
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Ramos F, Leonard J, Clemente-Blanco A, and Aragón L
- Subjects
- Chromatin genetics, DNA Packaging, Genome, Fungal, Mitosis, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae genetics, Adenosine Triphosphatases metabolism, Cell Cycle Proteins metabolism, Chromatin metabolism, Chromatin Immunoprecipitation methods, DNA-Binding Proteins metabolism, Multiprotein Complexes metabolism, Protein Tyrosine Phosphatases metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Chromosome condensation is an essential morphological event required for successful DNA segregation during mitosis. The high level of genome compaction achieved during this process is attained by the evolutionary conserved condensin complex. Recently, several lines of evidences have demonstrated that the mitotic phosphatase Cdc14 is required to ensure condensin loading onto chromosomes. To date several approaches have been used in order to characterize condensin activity and regulation, however these techniques are time-consuming and require complex equipment. In this chapter we described an easy and reliable protocol to analyze Cdc14-dependent condensin loading onto specific genomic DNA regions by using a chromatin immunoprecipitation (ChIP) technique.
- Published
- 2017
- Full Text
- View/download PDF
44. Comparative Analysis of piggyBac, CRISPR/Cas9 and TALEN Mediated BAC Transgenesis in the Zygote for the Generation of Humanized SIRPA Rats.
- Author
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Jung CJ, Ménoret S, Brusselle L, Tesson L, Usal C, Chenouard V, Remy S, Ouisse LH, Poirier N, Vanhove B, de Jong PJ, and Anegon I
- Subjects
- Animals, Humans, Mice, Mice, Transgenic, Rats, Rats, Transgenic, Antigens, Differentiation biosynthesis, Antigens, Differentiation genetics, CRISPR-Cas Systems, Chromosomes, Artificial, Bacterial genetics, Receptors, Immunologic biosynthesis, Receptors, Immunologic genetics, Transgenes, Zygote
- Abstract
BAC transgenic mammalian systems offer an important platform for recapitulating human gene expression and disease modeling. While the larger body mass, and greater genetic and physiologic similarity to humans render rats well suited for reproducing human immune diseases and evaluating therapeutic strategies, difficulties of generating BAC transgenic rats have hindered progress. Thus, an efficient method for BAC transgenesis in rats would be valuable. Immunodeficient mice carrying a human SIRPA transgene have previously been shown to support improved human cell hematopoiesis. Here, we have generated for the first time, human SIRPA BAC transgenic rats, for which the gene is faithfully expressed, functionally active, and germline transmissible. To do this, human SIRPA BAC was modified with elements to work in coordination with genome engineering technologies-piggyBac, CRISPR/Cas9 or TALEN. Our findings show that piggyBac transposition is a more efficient approach than the classical BAC transgenesis, resulting in complete BAC integration with predictable end sequences, thereby permitting precise assessment of the integration site. Neither CRISPR/Cas9 nor TALEN increased BAC transgenesis. Therefore, an efficient generation of human SIRPA transgenic rats using piggyBac opens opportunities for expansion of humanized transgenic rat models in the future to advance biomedical research and therapeutic applications.
- Published
- 2016
- Full Text
- View/download PDF
45. A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals: A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis.
- Author
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Chenouard V, Brusselle L, Heslan JM, Remy S, Ménoret S, Usal C, Ouisse LH, NGuyen TH, Anegon I, and Tesson L
- Subjects
- Animals, Electrophoresis, Capillary economics, Genotyping Techniques instrumentation, Mutation, Rats, Time Factors, Cost-Benefit Analysis, Electrophoresis, Capillary instrumentation, Gene Editing, Genotyping Techniques economics, Lab-On-A-Chip Devices
- Abstract
The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species. Moreover, these novel tools have become easier to use and have resulted in a great increase of applications. Whilst gene knockout (KO) or knockin (KI) animal models are relatively easy to achieve, there is a bottleneck in the detection and analysis of these mutations. Although several methods exist to detect these targeted mutations, we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process. The HMA-CE method uses a simple PCR amplification of genomic DNA (gDNA) followed by an automated capillary electrophoresis step which reveals a heteroduplexes (HD) signature for each mutation. This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level., (Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
46. Monitoring Chitin Deposition During Septum Assembly in Budding Yeast.
- Author
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Arcones I and Roncero C
- Subjects
- Chitin Synthase metabolism, Cell Wall metabolism, Chitin metabolism, Cytokinesis, Microscopy, Fluorescence methods, Saccharomycetales physiology
- Abstract
The synthesis of the septum is a critical step during cytokinesis in the fungal cell. Moreover, in Saccharomyces cerevisiae septum assembly depends mostly on the proper synthesis and deposition of chitin and, accordingly, on the timely regulation of chitin synthases. In this chapter, we will see how to follow chitin synthesis by two complementary approaches: monitoring chitin deposition in vivo at the septum by calcofluor staining and fluorescence microscopy, and measuring the chitin synthase activities responsible for this synthesis.
- Published
- 2016
- Full Text
- View/download PDF
47. Genome Editing in Rats Using TALE Nucleases.
- Author
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Tesson L, Remy S, Ménoret S, Usal C, Thinard R, Savignard C, De Cian A, Giovannangeli C, Concordet JP, and Anegon I
- Subjects
- Animals, Genome, Homologous Recombination genetics, Humans, RNA, Messenger genetics, Rats, Trans-Activators genetics, Animals, Genetically Modified genetics, Endonucleases genetics, Gene Knockout Techniques methods
- Abstract
The rat is an important animal model to understand gene function and model human diseases. Since recent years, the development of gene-specific nucleases has become important for generating new rat models of human diseases, to analyze the role of genes and to generate human antibodies. Transcription activator-like (TALE) nucleases efficiently create gene-specific knockout rats and lead to the possibility of gene targeting by homology-directed recombination (HDR) and generating knock-in rats. We describe a detailed protocol for generating knockout and knock-in rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.
- Published
- 2016
- Full Text
- View/download PDF
48. Transgenic animals and genetic engineering techniques. Nantes, France, 2-3 July, 2015.
- Author
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Ménoret S, Tesson L, Remy S, Usal C, Ouisse LH, Brusselle L, Chenouard V, Nguyen TH, David L, and Anegon I
- Subjects
- Animals, France, Research Report, Societies, Scientific, Time Factors, Animals, Genetically Modified genetics, Genetic Engineering methods
- Published
- 2015
- Full Text
- View/download PDF
49. Regulatory B Cells with a Partial Defect in CD40 Signaling and Overexpressing Granzyme B Transfer Allograft Tolerance in Rodents.
- Author
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Durand J, Huchet V, Merieau E, Usal C, Chesneau M, Remy S, Heslan M, Anegon I, Cuturi MC, Brouard S, and Chiffoleau E
- Subjects
- Allografts, Animals, Antigens, CD immunology, Cytokines immunology, Isoantigens immunology, Male, Rats, T-Lymphocytes, Regulatory immunology, B-Lymphocytes, Regulatory immunology, CD40 Antigens immunology, Granzymes immunology, Heart Transplantation, Plasma Cells immunology, Signal Transduction immunology, Transplantation Tolerance
- Abstract
Emerging knowledge regarding B cells in organ transplantation has demonstrated that these cells can no longer be taken as mere generators of deleterious Abs but can also act as beneficial players. We previously demonstrated in a rat model of cardiac allograft tolerance induced by short-term immunosuppression an accumulation in the blood of B cells overexpressing inhibitory molecules, a phenotype also observed in the blood of patients that spontaneously develop graft tolerance. In this study, we demonstrated the presence in the spleen of regulatory B cells enriched in the CD24(int)CD38(+)CD27(+)IgD(-)IgM(+/low) subpopulation, which are able to transfer donor-specific tolerance via IL-10 and TGF-β1-dependent mechanisms and to suppress in vitro TNF-α secretion. Following anti-CD40 stimulation, IgD(-)IgM(+/low) B cells were blocked in their plasma cell differentiation pathway, maintained high expression of the inhibitory molecules CD23 and Bank1, and upregulated Granzyme B and Irf4, two molecules described as highly expressed by regulatory B cells. Interestingly, these B cells recognized specifically a dominant donor Ag, suggesting restricted specificity that could lead to a particular B cell response. Regulatory B cells were not required for induction of tolerance and appeared following Foxp3(+)CD4(+)CD25(+) regulatory T cells, suggesting cooperation with regulatory T cells for their expansion. Nevertheless, following transfer to new recipients, these B cells migrated to the allograft, kept their regulatory profile, and promoted local accumulation of Foxp3(+)CD4(+)CD25(+) regulatory T cells. Mechanisms of regulatory B cells and their cell therapy potential are important to decipher in experimental models to pave the way for future developments in the clinic., (Copyright © 2015 by The American Association of Immunologists, Inc.)
- Published
- 2015
- Full Text
- View/download PDF
50. Homology-directed repair in rodent zygotes using Cas9 and TALEN engineered proteins.
- Author
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Ménoret S, De Cian A, Tesson L, Remy S, Usal C, Boulé JB, Boix C, Fontanière S, Crénéguy A, Nguyen TH, Brusselle L, Thinard R, Gauguier D, Concordet JP, Cherifi Y, Fraichard A, Giovannangeli C, and Anegon I
- Subjects
- Animals, Mice, Mice, Inbred C57BL, Microinjections, Rats, Rats, Sprague-Dawley, CRISPR-Cas Systems genetics, Protein Engineering, Recombinational DNA Repair genetics, Zygote physiology
- Abstract
The generation of genetically-modified organisms has been revolutionized by the development of new genome editing technologies based on the use of gene-specific nucleases, such as meganucleases, ZFNs, TALENs and CRISPRs-Cas9 systems. The most rapid and cost-effective way to generate genetically-modified animals is by microinjection of the nucleic acids encoding gene-specific nucleases into zygotes. However, the efficiency of the procedure can still be improved. In this work we aim to increase the efficiency of CRISPRs-Cas9 and TALENs homology-directed repair by using TALENs and Cas9 proteins, instead of mRNA, microinjected into rat and mouse zygotes along with long or short donor DNAs. We observed that Cas9 protein was more efficient at homology-directed repair than mRNA, while TALEN protein was less efficient than mRNA at inducing homology-directed repair. Our results indicate that the use of Cas9 protein could represent a simple and practical methodological alternative to Cas9 mRNA in the generation of genetically-modified rats and mice as well as probably some other mammals.
- Published
- 2015
- Full Text
- View/download PDF
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