Impaired Lymphocytes Development and Xenotransplantation of Gastrointestinal Tumor Cells in Prkdc-Null SCID Zebrafish Model.
Ontology highlight
ABSTRACT:
SUBMITTER: Jung IH
PROVIDER: S-EPMC5018095 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Cell transplantation into immunodeficient mice has revolutionized our understanding of regeneration, stem cell self-renewal, and cancer; yet models for direct imaging of engrafted cells has been limited. Here, we characterize zebrafish with mutations in recombination activating gene 2 (rag2), DNA-dependent protein kinase (prkdc), and janus kinase 3 (jak3). Histology, RNA sequencing, and single-cell transcriptional profiling of blood showed that rag2 hypomorphic mutant zebrafish lack T cells, whereas prkdc deficiency results in loss of mature T and B cells and jak3 in T and putative Natural Killer cells. Although all mutant lines engraft fluorescently labeled normal and malignant cells, only the prkdc mutant fish reproduced as homozygotes and also survived injury after cell transplantation. Engraftment into optically clear casper, prkdc-mutant zebrafish facilitated dynamic live cell imaging of muscle regeneration, repopulation of muscle stem cells within their endogenous niche, and muscle fiber fusion at single-cell resolution. Serial imaging approaches also uncovered stochasticity in fluorescently labeled leukemia regrowth after competitive cell transplantation into prkdc mutant fish, providing refined models to assess clonal dominance and progression in the zebrafish. Our experiments provide an optimized and facile transplantation model, the casper, prkdc mutant zebrafish, for efficient engraftment and direct visualization of fluorescently labeled normal and malignant cells at single-cell resolution.
| S-EPMC5110017 | biostudies-literature
Project description:The DNA-dependent protein kinase catalytic subunit (DNA-PKcs; encoded by PRKDC) functions in DNA non-homologous end-joining (NHEJ), the major DNA double strand break (DSB) rejoining pathway. NHEJ also functions during lymphocyte development, joining V(D)J recombination intermediates during antigen receptor gene assembly. Here, we describe a patient with compound heterozygous mutations in PRKDC, low DNA-PKcs expression, barely detectable DNA-PK kinase activity, and impaired DSB repair. In a heterologous expression system, we found that one of the PRKDC mutations inactivated DNA-PKcs, while the other resulted in dramatically diminished but detectable residual function. The patient suffered SCID with reduced or absent T and B cells, as predicted from PRKDC-deficient animal models. Unexpectedly, the patient was also dysmorphic; showed severe growth failure, microcephaly, and seizures; and had profound, globally impaired neurological function. MRI scans revealed microcephaly-associated cortical and hippocampal dysplasia and progressive atrophy over 2 years of life. These neurological features were markedly more severe than those observed in patients with deficiencies in other NHEJ proteins. Although loss of DNA-PKcs in mice, dogs, and horses was previously shown not to impair neuronal development, our findings demonstrate a stringent requirement for DNA-PKcs during human neuronal development and suggest that high DNA-PK protein expression is required to sustain efficient pre- and postnatal neurogenesis.
| S-EPMC3999051 | biostudies-literature
Project description:Here we report that a new nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse line harboring a complete null mutation of the common cytokine receptor gamma chain (NOD/SCID/interleukin 2 receptor [IL2r] gamma(null)) efficiently supports development of functional human hemato-lymphopoiesis. Purified human (h) CD34(+) or hCD34(+)hCD38(-) cord blood (CB) cells were transplanted into NOD/SCID/IL2rgamma(null) newborns via a facial vein. In all recipients injected with 10(5) hCD34(+) or 2 x 10(4) hCD34(+)hCD38(-) CB cells, human hematopoietic cells were reconstituted at approximately 70% of chimerisms. A high percentage of the human hematopoietic cell chimerism persisted for more than 24 weeks after transplantation, and hCD34(+) bone marrow grafts of primary recipients could reconstitute hematopoiesis in secondary NOD/SCID/IL2rgamma(null) recipients, suggesting that this system can support self-renewal of human hematopoietic stem cells. hCD34(+)hCD38(-) CB cells differentiated into mature blood cells, including myelomonocytes, dendritic cells, erythrocytes, platelets, and lymphocytes. Differentiation into each lineage occurred via developmental intermediates such as common lymphoid progenitors and common myeloid progenitors, recapitulating the steady-state human hematopoiesis. B cells underwent normal class switching, and produced antigen-specific immunoglobulins (Igs). T cells displayed the human leukocyte antigen (HLA)-dependent cytotoxic function. Furthermore, human IgA-secreting B cells were found in the intestinal mucosa, suggesting reconstitution of human mucosal immunity. Thus, the NOD/SCID/IL2rgamma(null) newborn system might be an important experimental model to study the human hemato-lymphoid system.
| S-EPMC1895228 | biostudies-literature
Project description:Despite 2 decades of research, no clear function for annexin A1 (AnxA1) has been established. Using AnxA1-KO mice, we show that tumor growth and metastasis are significantly decreased, whereas rodent survival and tumor necrosis are greatly increased when tumors grow in AnxA1-KO mice. Systems analysis of gene expression in these tumors specifically implicates 2 related vascular functions, angiogenesis and wound healing, in this impairment. Both tumor vascular development and wound healing are greatly retarded in KO tissues. Aortic ring assays reveal induced AnxA1 expression on sprouting endothelial cells of normal mice whereas KO aortas exhibit impaired endothelial cell sprouting that is rescued by adenoviral expression of AnxA1. Key differences in specific gene regulation may define new molecular pathways mediating angiogenesis, including a reset profile of pro- versus anti-angiogenic factors, apparently distinct for physiological versus pathological angiogenesis. These studies establish novel pro-angiogenic functions for AnxA1 in vascular endothelial cell sprouting, wound healing, and tumor growth and metastasis, thereby uncovering a new functional target for repairing damaged tissue and treating diseases such as cancer. They also provide critical new evidence that the tumor stroma and its microenvironment can greatly affect tumor progression and metastasis.
| S-EPMC2764877 | biostudies-literature
Project description:Lowe syndrome, which is characterized by defects in the central nervous system, eyes and kidneys, is caused by mutation of the phosphoinositide 5-phosphatase OCRL1. The mechanisms by which loss of OCRL1 leads to the phenotypic manifestations of Lowe syndrome are currently unclear, in part, owing to the lack of an animal model that recapitulates the disease phenotype. Here, we describe a zebrafish model for Lowe syndrome using stable and transient suppression of OCRL1 expression. Deficiency of OCRL1, which is enriched in the brain, leads to neurological defects similar to those reported in Lowe syndrome patients, namely increased susceptibility to heat-induced seizures and cystic brain lesions. In OCRL1-deficient embryos, Akt signalling is reduced and there is both increased apoptosis and reduced proliferation, most strikingly in the neural tissue. Rescue experiments indicate that catalytic activity and binding to the vesicle coat protein clathrin are essential for OCRL1 function in these processes. Our results indicate a novel role for OCRL1 in neural development, and support a model whereby dysregulation of phosphoinositide metabolism and clathrin-mediated membrane traffic leads to the neurological symptoms of Lowe syndrome.
| S-EPMC3313792 | biostudies-literature
Project description:Evidence for transgenerational inheritance of epigenetic information in vertebrates is scarce. Aberrant patterns of DNA methylation in gametes may set the stage for transmission into future generations. Here, we describe a viable hypomorphic allele of dnmt1 in zebrafish that causes widespread demethylation of CpG dinucleotides in sperm and somatic tissues. We find that homozygous mutants are essentially normal, with the exception of drastically impaired lymphopoiesis, affecting both larval and adult phases of T cell development. The phenotype of impaired larval (but not adult) T cell development is transmitted to subsequent generations by genotypically wildtype fish. We further find that about 200 differentially methylated regions in sperm DNA of transmitting and non-transmitting males, including hypermethylated sites associated with runx3 and rptor genes, whose reduced activities are associated with impaired larval T cell development. Our results indicate a particular sensitivity of larval T cell development to transgenerationally inherited epimutations.
| S-EPMC7481223 | biostudies-literature
Project description:The lack of preclinical models able to faithfully predict the immune responses which are later obtained in the clinic is a major hurdle for vaccines development as it increases markedly the delays and the costs required to perform clinical studies. We developed and evaluated the relevance to human immune responses of a novel humanized mouse model, humanized-spleen cells-NOD-SCID-gamma null (Hu-SPL-NSG), in which we grafted human spleen cells in immunodeficient NOD-SCID-IL-2r?null (NSG) mice. We selected the malaria vaccine candidate, Liver Stage Antigen 3-Full Length, because we had previously observed a major discrepancy between preclinical and clinical results, and compared its immunogenicity with that of a shorter form of the molecule, LSA3-729. NSG mice engrafted with human spleen lymphocytes were immunized with either LSA3-FL or LSA3-729, both adjuvanted with montanide ISA720. We found that the shorter LSA3-729 triggered the production of human antibodies and a T-helper-type 1 cellular immune response associated with protection whereas LSA3-FL did not. Results were consistent in five groups receiving lymphocytes from five distinct human donors. We identified antigenic regions in the full-length molecule, but not in the shorter version, which induced T-regulatory type of cellular responses. These regions had failed to be predicted by previous preclinical experiments in a wide range of animal models, including primates. Results were reproducible using spleen cells from all five human donors. The findings in the Hu-SPL-NSG model were similar to the results obtained using LSA3-FL in the clinic and hence could have been used to predict them. The model does not present graft versus host reaction, low survival of engrafted B lymphocytes and difficulty to raise primary immune responses, all limitations previously reported in humanized immune-compromised mice. Results also point to the shorter construct, LSA3-729 as a more efficient vaccine candidate. In summary, our findings indicate that the Hu-SPL-NSG model could be a relevant and cost-saving choice for early selection of vaccine candidates before clinical development, and deserves being further evaluated.
| S-EPMC5876497 | biostudies-literature
Project description:To study the influence of xenotransplantation on follicular recruitment and growth in cryopreserved/thawed human ovarian tissue.Two 3-mm pieces of cryopreserved/thawed human ovarian tissue obtained from female cancer patients (n?=?11) were xenotransplanted into a subcutaneous neck pouch of 6-week-old ovarectomized SCID mice (n?=?33) for 4 (n?=?18) and 12 (n?=?15) weeks.Thirty-two out of 33 mice survived the entire observation periods. Graft recovery rate was 95.58 % (65 of 68 grafts). The percentages of primordial follicles after 4 weeks (P?<?0.001) and 12 weeks (P?=?0.009) of grafting were significantly lower in comparison to pregraft controls. The percentage of secondary follicle was significantly higher after 4 weeks of grafting (P?=?0.018) and after 12 weeks (P?=?0.001) of grafting in comparison to pregraft controls. Ki67 immunohistochemistry showed that proliferative follicles were significantly higher after 4 and 12 weeks of grafting compared to pregraft controls (P?<?0.001). All follicles analyzed by TUNEL staining appeared healthy after xenotransplantation. The expression level of PTEN was reduced by 2.47-fold after 4 weeks of xenotransplantation, and this result was significant when 2-?Ct were analyzed (P?=?0.042).The higher proportion of growing follicles compared to resting follicles observed after xenotransplantation is most likely due to downregulation of PTEN gene expression followed by acceleration of follicular recruitment.
| S-EPMC5171895 | biostudies-other
Project description:BACKGROUND: Aberrant regulation of cell migration drives progression of many diseases, including cancer cell invasion and metastasis formation. Analysis of tumour invasion and metastasis in living organisms to date is cumbersome and involves difficult and time consuming investigative techniques. For primary human tumours we establish here a simple, fast, sensitive and cost-effective in vivo model to analyse tumour invasion and metastatic behaviour. METHODS: We fluorescently labelled small explants from gastrointestinal human tumours and investigated their metastatic behaviour after transplantation into zebrafish embryos and larvae. The transparency of the zebrafish embryos allows to follow invasion, migration and micrometastasis formation in real-time. High resolution imaging was achieved through laser scanning confocal microscopy of live zebrafish. RESULTS: In the transparent zebrafish embryos invasion, circulation of tumour cells in blood vessels, migration and micrometastasis formation can be followed in real-time. Xenografts of primary human tumours showed invasiveness and micrometastasis formation within 24 hours after transplantation, which was absent when non-tumour tissue was implanted. Furthermore, primary human tumour cells, when organotopically implanted in the zebrafish liver, demonstrated invasiveness and metastatic behaviour, whereas primary control cells remained in the liver. Pancreatic tumour cells showed no metastatic behaviour when injected into cloche mutant embryos, which lack a functional vasculature. CONCLUSION: Our results show that the zebrafish is a useful in vivo animal model for rapid analysis of invasion and metastatic behaviour of primary human tumour specimen.
| S-EPMC2697170 | biostudies-literature
Project description:Transplantation of human skin on immunodeficient mice that support engraftment with functional human immune systems would be an invaluable tool for investigating mechanisms involved in wound healing and transplantation. Nonobese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) readily engraft with human immune systems, but human skin graft integrity is poor. In contrast, human skin graft integrity is excellent on CB17-scid bg (SCID.bg) mice, but they engraft poorly with human immune systems.Human skin grafts transplanted onto immunodeficient NSG, SCID.bg, and other immunodeficient strains were evaluated for graft integrity, preservation of graft endothelium, and their ability to be rejected after engraftment of allogeneic peripheral blood mononuclear cells.Human skin transplanted onto NSG mice develops an inflammatory infiltrate, consisting predominately of host Gr1(+) cells, that is detrimental to the survival of human endothelium in the graft. Treatment of graft recipients with anti-Gr1 antibody reduces this cellular infiltrate, preserves graft endothelium, and promotes wound healing, tissue development, and graft remodeling. Excellent graft integrity of the transplanted skin includes multilayered stratified human epidermis, well-developed human vasculature, human fibroblasts, and passenger leukocytes. Injection of unfractionated, CD4 or CD8 allogeneic human peripheral blood mononuclear cell induces a rapid destruction of the transplanted skin graft.NSG mice treated with anti-Gr1 antibody provide a model optimized for both human skin graft integrity and engraftment of a functional human immune system. This model provides the opportunity to investigate mechanisms orchestrating inflammation, wound healing, revascularization, tissue remodeling, and allograft rejection and can provide guidance for improving outcomes after clinical transplantation.
| S-EPMC2901915 | biostudies-literature