Establishment of cell line and in vivo mouse model of canine Langerhans cell histiocytosis

2019 ◽  
Vol 17 (3) ◽  
pp. 345-353 ◽  
Author(s):  
Nguyen V. Son ◽  
Kazuyuki Uchida ◽  
Atigan Thongtharb ◽  
James K. Chambers ◽  
Takuya E. Kishimoto ◽  
...  
Keyword(s):  
Mouse Model ◽  
Cell Line ◽  
Langerhans Cell Histiocytosis ◽  
Langerhans Cell

Immunoliposomal Delivery of Mir-29b By Targeting Tumor Antigen ROR1 Induces Epigenetic Reprograming in Human-ROR1-Expressed Mouse Model of Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1743-1743
Author(s):  
Chi-Ling Chiang ◽  
Frank W Frissora ◽  
Zhiliang Xie ◽  
Xiaomeng Huang ◽  
Rajeswaran Mani ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Mouse Model ◽  
Cell Line ◽  
Tumor Antigen ◽  
Targeted Delivery ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Lymphocytic Leukemia

Abstract Chronic lymphocytic leukemia (CLL), characterized by accumulation of CD5+CD19+sIgM+ B lymphocytes in peripheral blood and lymphoid organs, is classified into indolent and aggressive forms. Patients with indolent CLL generally survive 5 to 10 years and do not require treatment until severe symptoms, while those with aggressive CLL show resistant to standard treatment and survive less than 24 months. While emerging B cell antigen receptor directed therapies are promising, resistance to such therapies pose problems warranting novel therapeutic approaches. MicroRNA (miR) profiling revealed lower expression of miR-29b in aggressive CLL associated with survival, drug resistance and poor prognosis via its up-regulation of anti-apoptotic proteins myeloid leukemia cell differentiation protein 1 (Mcl1) and oncogenic T-cell leukemia 1 (Tcl1). Thus, specific overexpression of miR-29b in B-CLL cells could be a potential therapy for aggressive CLL patients. Despite the promise, short circulation half-life, limited cellular uptake and off-target effects on non-desirable tissues pose a challenge for miR-based therapies. To promote efficiency and specificity of miR-29b delivery, we developed neutral immunonanoparticles with selectivity to CLL via targeting tumor antigen ROR1, which is expressed in over 95% of CLL but not normal B cells. We optimized a novel 2A2-immunoliposome (2A2-ILP) recognizing surface ROR1 on primary CLL cell to promote internalization and miR-29b uptake (n=6, p=0.042*). About 20-fold increased uptake of miR-29b was achieved with 2A2-ILP-miR-29b formulation compared to control. Further ROR1 targeted delivery of miR29b resulted in significant downregulation of DNMT1 and DNMT3a mRNA and protein (n=3, DNMT1: p= 0.0115*; DNMT3a: p=0.0231*, SP1; p=0.0031**) in primary CLL cells and a human CLL cell line OSU-CLL. Consistent with the downregulation of DNMTs, decreased global DNA methylation was observed in OSU-CLL cell line one week post- treatment with 2A2-ILP-miR-29b (n=3, p=0.0003***). To further study the in vivo ROR1-targeting efficiency of 2A2-ILP-miR-29b, we used our recently described Eµ-hROR1x Tcl1 CLL mouse model that develops CLL like disease with human ROR1 antigen in leukemic CD19+CD5+ B cells. Using hROR1+CD19+CD5+ leukemic cell engraftment model, we showed significant in-vivo efficacy of ROR1-ILP-miR-29b formulation associated with a) decreased number of circulating leukemic B220+CD5+ cells b) reduced splenomegaly (p=0.0461*, 2A2-29b: n=9; PBS: n=8) c) with extended survival (p=0.0075**, 2A2-29b: n=9; IgG-29b: n=7; 2A2-SC: n=7; PBS: n=8). In summary, 2A2-ILP effectively delivered functional miR-29b, resulting in downregulation of DNMT1 and DNMT3a, reduction of hypermethylation and anti-leukemic activity. Ongoing studies are aimed at understanding miR-29b mediated in-vivo methylome reprograming using our novel hROR1xTcl1 transgenic mouse model and ROR1-targeted miR-29b delivery formulation. Figure 1. Figure 1. Disclosures Byrd: Acerta Pharma BV: Research Funding.

scholarly journals Combination Therapy of a Host Defense-Like Lytic Peptide and Continuous Low-Dose Doxorubicin Inhibits Tumor Growth in a Syngeneic Immunocompetent Murine Fibrosarcoma (BFS-1) Model

2020 ◽  
Author(s):  
Jan Philip Suppelna ◽  
Kamran Harati ◽  
Andrea Rittig ◽  
Ingo Stricker ◽  
Markus Lehnhardt ◽  
...  
Keyword(s):  
Mouse Model ◽  
Tumor Growth ◽  
Cell Line ◽  
Host Defense ◽  
Low Dose ◽  
Sarcoma Cell ◽  
Fibrosarcoma Cell ◽  
Murine Fibrosarcoma

Abstract Background: The concept of a multimodality therapy in the treatment of soft tissue sarcomas (STS) has been discussed with controversy. Surgical resection with clear margins and radiation therapy remain gold standard in STS therapy. It is still questionable whether a systemic therapy with chemotherapeutics has a positive impact on the overall survival rate especially in early stages of disease, because the therapeutic effect in the treatment of STS is limited by its toxicities and its low responding rates. Treatment options are rare. As a result the search for combination therapies by using low dose approaches is of high importance. Recent studies showed the therapeutic efficiency of a designer host defense-like lytic D,L- amino acid peptide [D]-K 3 H 3 L 9 . Therefore we tested a combination of this peptide with Doxorubicin on two different sarcoma cell lines in vitro and also in a syngeneic immunocompetent murine fibrosarcoma mouse model. Methods: In vitro the human synovial sarcoma cell line SW 982 and the murine fibrosarcoma cell line BFS-1 were exposed to the oncolytic peptide [D]-K 3 H 3 L 9 , to the Anthracycline Doxorubicin and to both agents simultaneously. In vivo the murine fibrosarcoma cell line BFS-1 was injected subcutaneously into the syngeneic mice. When the tumors engrafted the oncolytic designer peptide [D]-K 3 H 3 L 9 , Doxorubicin or a combination of both was administered thrice a week for a three weeks’ follow-up. Results: The combination treatment approach using an oncolytic designer host defense peptide and Doxorubicin inhibited the in vitro sarcoma cell proliferation significantly. The single therapies, either with local intratumoral application of [D]-K 3 H 3 L 9 or with intraperitoneal application of Doxorubicin in the syngeneic mouse model, inhibited at least the tumor progression. The combination of both substances revealed a significant inhibition of tumor growth and weight. Conclusion: The in vivo low dose combination schedule inhibited the tumor growth significantly. Histological analyses of the tumor sections revealed an antiproliferative and antiangiogenic effect. So, these results demonstrate the effectiveness of combined low-dose application forms with designer host defense-like lytic peptides and chemotherapeutics.

Efficacy of NK-92 Against Human Multiple Myeloma in a NOD/SCID Gamma Null Mouse Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1854-1854
Author(s):  
Brenna E. Swift ◽  
Brent A. Williams ◽  
Yoko Kosaka ◽  
Joaquín Martínez-López ◽  
Xinghua Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Mouse Model ◽  
Disease Progression ◽  
Cell Line ◽  
Xenograft Model ◽  
Tumor Burden ◽  
Clinical Disease ◽  
Nsg Mice

Abstract Abstract 1854 Introduction: Human NK cell lines NK-92 and KHYG-1 exhibit cytotoxicity against a broad range of tumor types in vitro, including multiple myeloma (MM). To further test efficacy of the NK lines against MM, we developed a bioluminescent mouse model that recapitulates clinical MM using the human U266 MM cell line transduced to express GFP and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow (BM) engraftment. Results: In a pilot study in which 2×106 U266 cells were injected intravenously into NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice, CD138+ MM cells engrafted in BM, with no detectable engraftment in the liver, lungs, spleen, heart, or kidneys by anti-CD138 immunohistochemistry staining at 10 weeks. We used the U266eGFPluc bioluminescent NSG mouse model to evaluate efficacy of NK-92 cell therapy in vivo. We gave 10×106 NK-92 cells every 5 days to a total dose of 50×106 cells 7 days after MM injection. Tumor burden was monitored weekly by bioluminescence imaging 4 weeks after MM inoculation using the IVIS Imaging System, and LivingImage™ Software was used to acquire images and quantify bioluminescence. We showed that U266eGFPluc cells localized to BM and spine, reflecting MM pathophysiology. Disease burden in the NK-92 treated group was consistently lower than controls over time and significantly lower at 8 weeks (Dorsal and Ventral Mann-Whitney p=0.0381) whereas for KHYG-1, the signal increased slightly over control, but was not significant at 8 weeks (Mann-Whitney Dorsal p=0.540 and Ventral p=0.247). Clinical disease progression in MM control mice correlated with IVIS signal intensity at week 11 (r2=0.4; F test p=0.0279). Engraftment was determined by sacrificing mice at 10 weeks and analyzing BM for GFP+ cells by flow cytometry. Engraftment of MM cells in BM was as follows (mean+/− SEM): control (5 +/− 1.9%), NK-92 (0.24 +/− 0.19%) and KHYG-1 (5.2 +/− 1.6%) showing a trend toward a significant decrease in mean engraftment for the NK-92 group versus control (unpaired student's t test p=0.055), but not for KHYG-1 (p=0.939). One of 6 control mice had low engraftment with U266eGFPluc at 10 weeks increasing the variance of the control mean. There was a statistically significant decrease in median engraftment in the NK-92 group (Mann-Whitney p=0.019), but not for KHYG-1 (p =0.792) (Figure). GFP BM engraftment corresponded with bioluminescence detected in R and L BM by IVIS. Presence of NK cells in BM was detected in only 1/3 NK-92 mice tested (0.2%) and in none of the KHYG-1 mice at 10 weeks. To assess biodistribution of KHYG-1 we injected 10×106 CFSE-labeled KHYG-1 via tail vein into healthy NSG mice. Blood and organ samples were collected 8 and 24 hours later and analyzed by flow cytometry. We detected CFSE-labeled KHYG-1 primarily in liver, blood and lung, less in kidney, and none in heart, spleen or BM. Conclusion: We have established a human MM cell line xenograft model in NSG mice comparable to clinical disease. Treatment efficacy can be monitored in live NSG mice by IVIS imaging technology and tumor burden at sacrifice can be determined by GFP detection. MM progression was reduced by NK-92, but not KHYG-1 as measured by bioluminescence and reduction of engrafted U266eGFPluc cells. We have shown that a MM xenograft model can screen for in vivo efficacy of immune therapies for MM. Our results indicate that NK-92 is a potentially effective therapy for MM. Disclosures: No relevant conflicts of interest to declare.

Successful in vivo immunolocalization of Langerhans cell histiocytosis with use of a monoclonal antibody, NA1/34

1994 ◽  
Vol 125 (5) ◽  
pp. 717-722 ◽  
Author(s):  
K.M. Kelly ◽  
P.C.L. Beverley ◽  
A.C. Chu ◽  
V. Davenport ◽  
I. Gordon ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Langerhans Cell Histiocytosis ◽  
Langerhans Cell

DOR-1, A novel CD10+ stromal cell line derived from progressive Langerhans cell histiocytosis of bone

2005 ◽  
Vol 44 (2) ◽  
pp. 128-137 ◽  
Author(s):  
Jean Gogusev ◽  
Louise Telvi ◽  
Ichiro Murakami ◽  
Yves Lepelletier ◽  
Christian Nezelof ◽  
...  
Keyword(s):  
Cell Line ◽  
Langerhans Cell Histiocytosis ◽  
Stromal Cell ◽  
Langerhans Cell ◽  
Stromal Cell Line

scholarly journals Establishment of a Langerhans cell histiocytosis lesion cell line with dermal dendritic cell characteristics

2014 ◽  
Vol 33 (1) ◽  
pp. 171-178 ◽  
Author(s):  
ICHIRO MURAKAMI ◽  
JEAN GOGUSEV ◽  
FRANCIS JAUBERT ◽  
MICHIKO MATSUSHITA ◽  
KAZUHIKO HAYASHI ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Cell Line ◽  
Langerhans Cell Histiocytosis ◽  
Langerhans Cell ◽  
Dermal Dendritic Cell

scholarly journals Case Report: Characterizing the Role of the STXBP2-R190C Monoallelic Mutation Found in a Patient With Hemophagocytic Syndrome and Langerhans Cell Histiocytosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Laura Viñas-Giménez ◽  
Rafael Rincón ◽  
Roger Colobran ◽  
Xavier de la Cruz ◽  
Verónica Paola Celis ◽  
...  
Keyword(s):  
Cell Line ◽  
Langerhans Cell Histiocytosis ◽  
Nk Cell ◽  
Adaptive Immune System ◽  
Langerhans Cell ◽  
Adaptive Immune ◽  
Life Threatening ◽  
Monoallelic Mutation ◽  
Nk Cell Cytotoxicity

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory disorder. HLH can be considered as a threshold disease depending on the trigger and the residual NK-cell cytotoxicity. In this study, we analyzed the molecular and functional impact of a novel monoallelic mutation found in a patient with two episodes of HLH. A 9-month-old child was diagnosed at 2 months of age with cutaneous Langerhans cell histiocytosis (LCH). After successful treatment, the patient developed an HLH episode. At 16 month of age, the patient went through an HSCT losing the engraftment 5 months later concomitant with an HLH relapse. The genetic study revealed a monoallelic mutation in the STXBP2 gene (.pArg190Cys). We transfected COS7 cells to analyze the STXBP2-R190C expression and to test the interaction with STX11. We used the RBL-2H3 cell line expressing STXBP2-WT-EGFP or R190C-EGFP for degranulation assays. Mutation STXBP2-R190C did not affect protein expression or interaction with syntaxin-11. However, we have demonstrated that STXBP2-R190C mutation diminishes degranulation in the RBL-2H3 cell line compared with the RBL-2H3 cell line transfected with STXBP2-WT or nontransfected. These results suggest that STXBP2-R190C mutation acts as a modifier of the degranulation process producing a decrease in degranulation. Therefore, under homeostatic conditions, the presence of one copy of STXBP2-R190 could generate sufficient degranulation capacity. However, it is likely that early in life when adaptive immune system functions are not sufficiently developed, an infection may not be resolved with this genetic background, leading to a hyperinflammation syndrome and eventually develop HLH. This analysis highlights the need for functional testing of new mutations to validate their role in genetic susceptibility and to establish the best possible treatment for these patients.

scholarly journals Creating and Testing a CD207+ Langerhans Cell Histiocytosis-like Cell Line

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5399-5399
Author(s):  
Jessica Velazquez ◽  
Amel Sengal ◽  
Carl E. Allen ◽  
Rikhia Chakraborty
Keyword(s):  
Cell Line ◽  
Langerhans Cell Histiocytosis ◽  
Conflicts Of Interest ◽  
Mapk Pathway ◽  
Langerhans Cell ◽  
Mitogen Activated Protein Kinase ◽  
Established Cell Line ◽  
Human Telomerase Reverse Transcriptase ◽  
Cytokine Cocktail ◽  
Immortalized Cells

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by pathological CD207+ dendritic cells (DCs) with persistent mitogen-activated protein kinase (MAPK) activation. Investigations into LCH have historically been challenged by the small percentage of pathologic CD207+ DCs. No cell lines with morphology or function representing LCH lesion CD207+ DCs currently exist. We utilized four strategies to generate cell line(s) mimicking differentiated LCH pathogenic cells. First, CD207+ cells were isolated from the lesion of an LCH patient and cultured in a cytokine cocktail. The cells maintained CD1a and CD207 expression for two weeks, after which there were significant changes in cell morphology and progression to cell death. When a similar approach was implemented to isolate and culture skin CD207+ cells, the cells were viable for only three days, supporting the potential role for somatic activating MAPK mutations in LCH lesion DCs to prolong viability. CD207+ cells were then isolated from HLA-DR+ (lineage negative) cells from the lymphoid stroma of healthy tonsils (tCD207+). The tCD207+ cells were transduced with a lentivirus encoding human telomerase reverse transcriptase (hTERT). These cells, also lacking MAPK activating mutations, died two weeks post-transduction. A fourth approach has been more successful in which tCD207+ cells were cultured in a cytokine cocktail which provided MAPK pathway stimulation. The cells retained CD207 expression and survived in culture for over two weeks. The cells were then immortalized using a lentivirus encoding HOXA9. Immortalized cells maintained CD207 expression. Allele specific MAPK pathway mutations (BRAF and MAP2K1) are being generated by class II CRISPR/Cpf1 genome editing as Cpf1 has been shown to have robust activity to induce specific disruption of only mutant, but not wild-type, BRAF allele. The phenotypic and genomic characteristics of the immortalized cells expressing the different MAPK pathway mutations will be assessed by RHG-banding cytogenetic analysis, fluorescence in situ hybridization, gene expression analysis, and immuno‐cytochemistry and results will be compared to cells isolated from LCH lesions to confirm whether the established cell line may be a viable in vitro mimic of the LCH lesion DC. Disclosures No relevant conflicts of interest to declare.

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