An effective method for establishing human B lymphoblastic cell lines using epstein-barr virus

Caputo, Jane; Thompson, Ariane A.; McClintock, Patrick R.; Reid, Yvonne; Hay, Robert J.

doi:10.1007/bf02388202

Cited by 37 publications

(18 citation statements)

References 7 publications

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“…The buffy coat was washed twice in PBS and subjected to fusion with myeloma cells. EBV transformation of patient B cells was conducted by the University of North Carolina Tissue Culture Facility by standard procedures (30). Fused heterohybridomas were cultured in RPMI 1640 with 20% FCS and hybridoma-cloning factors (BioVeris) plus hypoxanthine aminopterin thymidine and 10 M ouabain in 96-well plates.…”

Section: Cell Preparation and Cell Fusionmentioning

confidence: 99%

Dissecting the Anti-Desmoglein Autoreactive B Cell Repertoire in Pemphigus Vulgaris Patients

Qian

Díaz

et al. 2007

The Journal of Immunology

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Pemphigus vulgaris (PV) encompasses two clinical phenotypes, one producing mucosal blisters and the other mucosal and skin lesions (mcPV). The mucosal blister-producing PV variant is characterized by autoantibodies against desmoglein (Dsg)3, whereas mucosal and skin lesion-producing PV is characterized by autoantibodies to Dsg3 and Dsg1. The present study was aimed at disclosing the diversity and clonality of the anti-Dsg3 response, as well as whether anti-Dsg3 B cells are Ag selected. Human-mouse heterohybridomas were generated by fusion of EBV-transformed or freshly isolated PBLs from six PV patients with mouse myeloma cells. A total of 73 anti-Dsg hybridomas (47 IgM and 26 IgG) were isolated. Over 90% are specific for both Dsg1 and Dsg3 indicating extensive cross-reactivity between these responses. VH gene segment use by IgM hybridomas is diverse, but is restricted among IgG hybridomas, where the majority uses one of two VH genes. VL gene segment use was diverse even among IgG hybridomas suggesting that the VL is less critical to defining desmoglein specificity. Additionally, the IgG hybridomas were extensively mutated and the distribution and nature of the mutations suggested that they had been Ag selected. We conclude that the potentially pathogenic IgG anti-Dsg response is restricted in VH use, is somatically mutated, and is Ag selected.

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Section: Cell Preparation and Cell Fusionmentioning

confidence: 99%

Dissecting the Anti-Desmoglein Autoreactive B Cell Repertoire in Pemphigus Vulgaris Patients

Qian

Díaz

et al. 2007

The Journal of Immunology

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“…Blood samples from members of family IGG-E were collected, lymphocytes EBV-transformed [20] and DNA isolated according to standard protocols [21] . DNA was also extracted from surgically resected tumor samples.…”

Section: Dna Isolationmentioning

confidence: 99%

Genetic Predisposition to Familial Neuroblastoma: Identification of Two Novel Genomic Regions at 2p and 12p

et al. 2007

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Objectives: The rarity of familial neuroblastoma (NB) has allowed only a few linkage studies, most of which did not show any evidence of linkage to regions involved in somatic alterations or to genes implicated in other neurocristopathies seldom associated with NB. We screened a highly informative family with recurrent NB by genome-wide linkage analysis aimed at identifying chromosomal regions for NB predisposing genes. Methods: A genome-wide screen was performed using 382 microsatellite markers. Multipoint model-based linkage analysis was carried out under a dominant mode of inheritance for the disease using the ‘affected only’ approach. Results: Our analysis identified two haplotypes co-segregating with the disease on chromosomes 2p and 12p, and yielded maximum lod-score values of 3.01 (p < 0.0001) for markers on both intervals. Conclusions: Evidence of linkage was reported at 16p in North American families, whereas our studies excluded this interval and indicated other loci for disease predisposition, thus confirming the remarkable genetic heterogeneity of NB. These results suggest an oligogenic inheritance in NB involving more loci in genetic determination of the disease.

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“…EBV generation procedures were undertaken according to the provider's protocol and their published methods. 8 …”

Section: Cells and Epstein-barr Virusmentioning

confidence: 97%

Stimulation of Epstein-Barr virus-infected human B cell growth by physiological concentrations of 4-hydroxynonenal

Ranjan

Chen

Johnston

et al. 2006

Cell Biochem. Funct.

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We had previously shown that cyclosporin A (CsA) directly promoted the immortalization of Epstein-Barr virus (EBV)-infected human B cells (EBV-B cells) via an oxidative stress mechanism. 4-Hydroxynonenal (HNE) is a reactive end-product of lipid peroxidation. We hypothesized that HNE may mediate a direct oxidative stress-promoting effect of CsA on EBV-B cells. HNE-protein adducts in CsA-treated EBV-B cell extracts were assayed immunochemically using a Slot-Blot method. Cell proliferation was assayed by [(3)H]-thymidine incorporation. EBV oncogene latent membrane protein-1 (LMP1) expression was assayed by using PE-conjugated anti-LMP1 antibody in flow cytometry. We found that CsA at 500 ng ml(-1) and 1000 ng ml(-1) significantly increased the level of HNE-protein adducts in EBV-B cells over the control (arbitrary units +/- SE) by 251.3 +/- 7.5 to 361.3 +/- 9.7 and 342.7 +/- 10.7, respectively (p < 0.05, n = 3). EBV-B cells treated with a physiological concentration of HNE (1 microM) for 0.5 and 1 h and cultured for 2 and 4 weeks showed significantly increased [(3)H]-thymidine incorporation. EBV-B cells treated with HNE (1 microM) for 1 h and subsequently cultured for 2 and 4 weeks had a significantly higher ( > 2.0 times) LMP1-positive cell population over the control. In conclusion, in accordance with our previous findings, we show that CsA treatment of EBV-B cells results in increased production of the lipid peroxidation reactive end-product HNE that directly promotes EBV-B cell proliferation and LMP1 expression. This observation provides evidence for further understanding the mechanism of CsA-induced oxidative stress on EBV-related post-transplant lymphoproliferative disorder (PTLD).

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An effective method for establishing human B lymphoblastic cell lines using epstein-barr virus

Cited by 37 publications

References 7 publications

Dissecting the Anti-Desmoglein Autoreactive B Cell Repertoire in Pemphigus Vulgaris Patients

Dissecting the Anti-Desmoglein Autoreactive B Cell Repertoire in Pemphigus Vulgaris Patients

Genetic Predisposition to Familial Neuroblastoma: Identification of Two Novel Genomic Regions at 2p and 12p

Stimulation of Epstein-Barr virus-infected human B cell growth by physiological concentrations of 4-hydroxynonenal

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