scholarly journals Transformation-associated alterations in interactions between pre-B cells and fibronectin

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2311-2320 ◽  
Author(s):  
FM Lemoine ◽  
S Dedhar ◽  
GM Lima ◽  
CJ Eaves
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cell Attachment ◽  
Attachment Site ◽  
Receptor Antibodies

Abstract Marrow stromal elements produce as yet uncharacterized soluble growth factors that can stimulate the proliferation of murine pre-B cells, although close contact between these two cell types appears to ensure a better pre-B cell response. We have now shown that freshly isolated normal pre-B cells (ie, the B220+, surface mu- fraction of adult mouse bone marrow) adhere to fibronectin (FN) via an RGD cell-attachment site, as shown in a serum-free adherence assay, and they lose this functional ability on differentiation in vivo into B cells (ie, the B220+, surface mu+ fraction). Similarly, cells from an immortalized but stromal cell-dependent and nontumorigenic murine pre-B cell line originally derived from a Whitlock-Witte culture were also found to adhere to fibronectin (FN) via an RGD cell-attachment site. Moreover, in the presence of anti-FN receptor antibodies, the ability of this immortalized pre-B cell line to proliferate when co-cultured with a supportive stromal cell line (M2–10B4 cells) was markedly reduced (down to 30% of control). This suggests that pre-B cell attachment to FN on stromal cells may be an important component of the mechanism by which stromal cells stimulate normal pre-B cell proliferation and one that is no longer operative to control their more differentiated progeny. Two differently transformed pre-B cell lines, both of which are autocrine, stromal-independent, tumorigenic in vivo, and partially or completely differentiation-arrested at a very early stage of pre-B cell development, did not bind to FN. In addition, anti-FN receptor antibodies were much less effective in diminishing the ability of these tumorigenic pre-B cells to respond to M2–10B4 cell stimulation, which could still be demonstrated when the tumorigenic pre-B cells were co- cultured with M2–10B4 cells at a sufficiently low cell density. Analysis of cell surface molecules immunoprecipitated from both the nontumorigenic and tumorigenic pre-B cell lines by an anti-FN receptor antibody showed an increase in very late antigen (VLA) alpha chain(s) in both tumorigenic pre-B cell lines and a decrease in the beta 1 chain in one. Interestingly, all of the pre-B cell lines expressed similar amounts of messenger RNA for the beta 1 chain of the FN receptor. These results suggest that alteration of FN receptor expression on pre-B cells may represent a mechanism contributing to the outgrowth of leukemic pre-B cells with an autocrine phenotype and capable of stromal cell-independent, autonomous growth.

scholarly journals Transformation-associated alterations in interactions between pre-B cells and fibronectin

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2311-2320 ◽  
Author(s):  
FM Lemoine ◽  
S Dedhar ◽  
GM Lima ◽  
CJ Eaves
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cell Attachment ◽  
Attachment Site ◽  
Receptor Antibodies

Marrow stromal elements produce as yet uncharacterized soluble growth factors that can stimulate the proliferation of murine pre-B cells, although close contact between these two cell types appears to ensure a better pre-B cell response. We have now shown that freshly isolated normal pre-B cells (ie, the B220+, surface mu- fraction of adult mouse bone marrow) adhere to fibronectin (FN) via an RGD cell-attachment site, as shown in a serum-free adherence assay, and they lose this functional ability on differentiation in vivo into B cells (ie, the B220+, surface mu+ fraction). Similarly, cells from an immortalized but stromal cell-dependent and nontumorigenic murine pre-B cell line originally derived from a Whitlock-Witte culture were also found to adhere to fibronectin (FN) via an RGD cell-attachment site. Moreover, in the presence of anti-FN receptor antibodies, the ability of this immortalized pre-B cell line to proliferate when co-cultured with a supportive stromal cell line (M2–10B4 cells) was markedly reduced (down to 30% of control). This suggests that pre-B cell attachment to FN on stromal cells may be an important component of the mechanism by which stromal cells stimulate normal pre-B cell proliferation and one that is no longer operative to control their more differentiated progeny. Two differently transformed pre-B cell lines, both of which are autocrine, stromal-independent, tumorigenic in vivo, and partially or completely differentiation-arrested at a very early stage of pre-B cell development, did not bind to FN. In addition, anti-FN receptor antibodies were much less effective in diminishing the ability of these tumorigenic pre-B cells to respond to M2–10B4 cell stimulation, which could still be demonstrated when the tumorigenic pre-B cells were co- cultured with M2–10B4 cells at a sufficiently low cell density. Analysis of cell surface molecules immunoprecipitated from both the nontumorigenic and tumorigenic pre-B cell lines by an anti-FN receptor antibody showed an increase in very late antigen (VLA) alpha chain(s) in both tumorigenic pre-B cell lines and a decrease in the beta 1 chain in one. Interestingly, all of the pre-B cell lines expressed similar amounts of messenger RNA for the beta 1 chain of the FN receptor. These results suggest that alteration of FN receptor expression on pre-B cells may represent a mechanism contributing to the outgrowth of leukemic pre-B cells with an autocrine phenotype and capable of stromal cell-independent, autonomous growth.

Genetic and Functional Characterization of Isolated Stromal Cell Lines from the Aorta-Gonado-Mesonephros (AGM)-Region.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2328-2328
Author(s):  
Katja C. Weisel ◽  
Ying Gao ◽  
Jae-Hung Shieh ◽  
Lothar Kanz ◽  
Malcolm A.S. Moore
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Stromal Cell Line ◽  
Mes Cells

Abstract The aorta-gonads-mesonephros (AGM) region autonomously generates adult repopulating hematopoietic stem cells (HSC) in the mouse embryo and provides its own HSC-supportive microenvironment. Stromal cells from adult bone marrow, yolk sac, fetal liver and AGM have been used in coculture systems for analysing growth, maintenance and differentiation of hematopoietic stem cells. We generated >100 cloned stromal cell lines from the AGM of 10.5 dpc mouse embryos. In previous studies, we tested these for support of murine adult and human cord blood (CB) CD34+ cells. We could demonstrate that 25 clones were superior to the MS5 bone marrow stromal cell line in supporting progenitor cell expansion of adult mouse bone marrow both, in 2ndry CFC and CAFC production. In addition we demonstrated that 5 AGM lines promoted in absence of exogenous growth factors the expansion of human CB cells with progenitor (CFC production for at least 5 weeks) and stem cell (repopulation of cocultured cells in NOD/SCID assay) function. Now, we could show that one of the isolated stromal cell lines (AGM-S62) is capable in differentiating undifferentiated murine embryonic stem (mES) cells into cells of the hematopoietic lineage. A sequential coculture of mES-cells with AGM-S62 showed production of CD41+ hematopoietic progenitor cells at day 10 as well as 2ndry CFC and CAFC production of day 10 suspension cells. Hematopoietic cell differentiation was comparable to standard OP9 differentiation assay. With these data, we can describe for the first time, that a stromal cell line other than OP9 can induce hematopoietic differentiation of undifferentiated mES cells. Hematopoietic support occurs independently of M-CSF deficiency, which is the characteristic of OP9 cells, because it is strongly expressed by AGM-S62. To evaluate genes responsible for hematopoietic cell support, we compared a supporting and a non-supporting AGM stromal cell line by microarray analysis. The cell line with hematopoietic support clearly showed a high expression of mesenchymal markers (laminins, thrombospondin-1) as well as characteristic genes for the early vascular smooth muscle phenotype (Eda). Both phenotypes are described for stromal cells with hematopoietic support generated from bone marrow and fetal liver. In addition, the analysed supporting AGM stromal cell line interestingly expressed genes important in early B-cell differentiation (osteoprotegerin, early B-cell factor 1, B-cell stimulating factor 3), which goes in line with data demonstrating early B-cell development in the AGM-region before etablishing of fetal liver hematopoiesis. Further studies will show the significance of single factors found to be expressed in microarray analyses. This unique source of > 100 various cell lines will be of value in elucidating the molecular mechanisms regulating embryonic and adult hematopoiesis in mouse and man.

scholarly journals Support of early B-cell differentiation in mouse fetal liver by stromal cells and interleukin-7

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2612-2617 ◽  
Author(s):  
Y Gunji ◽  
T Sudo ◽  
J Suda ◽  
Y Yamaguchi ◽  
H Nakauchi ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Liver Cells ◽  
B Cell Differentiation ◽  
Stromal Cell Line ◽  
Fetal Liver Cells

We compared the development of B-cell progenitors with that of myeloid progenitors in fetal liver cells at various gestational ages. Day 12 to 14 fetal liver cells did not form pre-B-cell colonies. Pre-B-cell colonies were developed from day 15 fetal liver cells. The incidence of colonies increased with increases in gestational age and reached a maximum on days 18 to 19. In contrast, the incidence of myeloid colonies formed in the presence of interleukin-3 (IL-3) and erythropoietin did not change significantly during days 13 to 21 of gestation. After coculturing day 13 fetal liver cells with IL-7- producing stromal cell line ST-2, they could respond to IL-7 and proliferate. Analysis of the phenotypes showed that day 13 fetal liver cells were B220-, IgM-, while culturing day 13 fetal liver cells with ST-2 and untreated day 18 fetal liver cells contained the population of B220+ cells. Even in the presence of IL-7-defective stromal cell line FLS-3, IL-7-responsive cells could be induced from day 13 fetal liver cells. IL-7 acted on B220+ cells and induced pre-B-cell colonies that contained IgM+ cells in the methylcellulose culture. IL-7 mRNA was expressed in days 13 and 18 fetal liver cells but not in pre-B cells or adult liver cells. From these findings, it is suggested that stromal cells or stromal-derived factors but not IL-7 were required for the differentiation from B220- cells to B220+ cells. In the second stage, B220+, IgM- cells proliferated and some of them differentiated to IgM+ cells in the presence of IL-7 alone. The two-step model can apply to in vivo early B lymphopoiesis.

scholarly journals Support of early B-cell differentiation in mouse fetal liver by stromal cells and interleukin-7

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2612-2617 ◽  
Author(s):  
Y Gunji ◽  
T Sudo ◽  
J Suda ◽  
Y Yamaguchi ◽  
H Nakauchi ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Liver Cells ◽  
B Cell Differentiation ◽  
Stromal Cell Line ◽  
Fetal Liver Cells

Abstract We compared the development of B-cell progenitors with that of myeloid progenitors in fetal liver cells at various gestational ages. Day 12 to 14 fetal liver cells did not form pre-B-cell colonies. Pre-B-cell colonies were developed from day 15 fetal liver cells. The incidence of colonies increased with increases in gestational age and reached a maximum on days 18 to 19. In contrast, the incidence of myeloid colonies formed in the presence of interleukin-3 (IL-3) and erythropoietin did not change significantly during days 13 to 21 of gestation. After coculturing day 13 fetal liver cells with IL-7- producing stromal cell line ST-2, they could respond to IL-7 and proliferate. Analysis of the phenotypes showed that day 13 fetal liver cells were B220-, IgM-, while culturing day 13 fetal liver cells with ST-2 and untreated day 18 fetal liver cells contained the population of B220+ cells. Even in the presence of IL-7-defective stromal cell line FLS-3, IL-7-responsive cells could be induced from day 13 fetal liver cells. IL-7 acted on B220+ cells and induced pre-B-cell colonies that contained IgM+ cells in the methylcellulose culture. IL-7 mRNA was expressed in days 13 and 18 fetal liver cells but not in pre-B cells or adult liver cells. From these findings, it is suggested that stromal cells or stromal-derived factors but not IL-7 were required for the differentiation from B220- cells to B220+ cells. In the second stage, B220+, IgM- cells proliferated and some of them differentiated to IgM+ cells in the presence of IL-7 alone. The two-step model can apply to in vivo early B lymphopoiesis.

Interactions with the Microenvironment Protect Lymphoma B-Cells From Rituximab Induced Apoptosis and Could Represent a Therapeutic Target

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3115-3115
Author(s):  
Marek Mraz ◽  
Clive S. Zent ◽  
Amy K Church ◽  
Nisar A Baig ◽  
Diane F. Jelinek ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Adhesion ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Stromal Cells ◽  
Research Funding ◽  
Stromal Cell ◽  
Plastic Surface ◽  
Induced Apoptosis

Abstract Abstract 3115 Background. Rituximab significantly improves the outcome of patients with B-cell non-Hodgkin lymphomas, but it does not completely eradicate residual B-cell populations in the bone marrow (BM) and lymph nodes (LN). The architecture and gene expression profile of LN stromal cells in diffuse large cell lymphoma correlates with outcome following treatment with R-CHOP (Lenz et al. 2008). Interestingly, multiple studies have demonstrated the importance of B-cells adhesion and pro-survival signaling mediated by integrin alfa-4-beta-1 (VLA-4/CD49d), which is constitutively expressed on malignant B-cells. Although several studies have demonstrated that adhesion to stromal cell cultures or ligand coated surfaces can protect malignant B-cells from apoptosis induced by chemotherapy drugs (cell adhesion-mediated drug resistance (CAM-DR); Dalton, 2002), a similar mechanism of resistance to rituximab has not, to our knowledge, been described. Hypothesis. In this study we tested the hypothesis that interactions with the microenvironment protect malignant B-cells from rituximab induced apoptosis, and that targeting these interactions with natalizumab, a humanized monoclonal anti-alfa-4 antibody approved for treatment of multiple sclerosis and Crohn's disease, can overcome this protection. Methods. Lymphoma B-cell lines were cultured on either a plastic surface, confluent HS-5 cells or a fibronectin (alfa-4-beta-1 ligand) coated surface. HS-5 is an immortalized human bone marrow stromal cell line and a well characterized model for a component of the BM microenvironment (Roecklein and Torok-Storb, 1995). Cells were treated with rituximab (10 ug/mL; Genentech), natalizumab (10ug/mL; Biogen IDEC), control IgG (10ug/mL, Sigma-Aldrich). Cell viability was measured by flow cytometry using Annexin V-FITC, propidium iodide in malignant B-cell population (anti-CD19 antibody, BD PharMingen). Results. We studied ten CD20-positive B-cell lymphoma cell lines and the CD20-positive MEC-1 cell line (derived from prolymfocytoid transformation of CLL) for rituximab induced apoptosis. The four most rituximab sensitive cell lines were used for further experiments (Karpas-422, Raji, DOHH2, SUDHL-4). Cell lines were co-cultured for 24hrs with confluent HS-5 stromal cells and subsequently treated for 24hrs with rituximab. The percentage of apoptotic cells was significantly lower (17-23% vs. 30–42%, p<0.05) for cells co-cultured with HS-5 cells compared to control cells cultivated under the same conditions on the plastic surface for all four cell lines. Similar results were obtained for several primary CLL samples (3-5% vs. 13–18%, p<0.05). We next examined if natalizumab could disrupt cell adhesion to fibronectin and overcome the stromal cell-mediated resistance to rituximab. Natalizumab reduced the number of lymphoma cells that adhered to fibronectin by 75–95% (p<0.05). Moreover, adherent cells cultivated on fibronectin coated surface completely lost the adhesion morphology after the addition of natalizumab to the cultivation media. The combination of natalizumab and rituximab versus rituximab alone increased by 26–32% (p<0.05) the number of apoptotic B-cells in co-culture with HS-5 for three of four rituximab-responding cell lines. Conclusion. We have shown that human bone marrow stromal cells (HS-5) protect lymphoma B cells from rituximab induced apoptosis suggesting existence of stromal cell adhesion-mediated antibody resistance (CAM-AR) analogous to CAM-DR. Targeting integrin alfa-4-beta-1 with natalizumab partially overcomes this cell adhesion-mediated resistance to rituximab. Research supported by P50CA97274-8 LYMPHOMA SPORE, Genentech, MSMT-MSM0021622430 and IGAMZCR NT11218-3/2010. Disclosures: Zent: Genentech: Research Funding; Genzyme: Research Funding; Novartis: Research Funding.

scholarly journals Stromal-Cell and Cytokine-Dependent Lymphocyte Clones Which Span the Pre-B- to B-Cell Transition

1991 ◽  
Vol 1 (3) ◽  
pp. 149-161 ◽  
Author(s):  
Katsuhiko Ishihara ◽  
Kay Medina ◽  
Shin-Ichi Hayashi ◽  
Carolynn Pietrangeli ◽  
Anthony E. Namen ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Light Chain ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cell Clone ◽  
Chain Complex ◽  
Immunodeficient Mice ◽  
Surrogate Light Chain

Five stromal-cell-dependent lymphocyte clones are described that correspond to late pre-B or early B-cell stages of differentiation.They are useful for determining the molecular requirements for pre-B replication, for studying the stromal cells that supply those factors, and for delineating the final sequence of differentiation events as newly formed lymphocytes prepare to exit the bone marrow. The efficiency of lymphocyte growth at limiting dilution varied substantially on different stromal-cell clones and may reflect functional heterogeneity of stromal cells. Most lymphocyte clones were similar to uncloned lymphocytes from Whitlock-Witte cultures in that they responded only transiently to interleukin-7 (IL-7) and then died, unless maintained on a stromal-cell clone. One unusual lymphocyte clone (2E8) was propagated for more than 1 year in IL-7 alone and was selectively responsive to that cytokine. Most of the lymphocyte clones were not tumorigenic in immunodeficient mice. However, one pre-B clone (1A9)’grew autonomously in culture when held at high density, responded to conditioned medium from a number of cell lines, and was tumorigenic. Tumors derived from this clone were infiltrated by stromal cells and lymphocytes taken from the tumors' retained characteristics of the original clone. Ly-6 antigens were inducible on 2E8 and 1A9 cells, but the lymphocytes were otherwise arrested in differentiation. The 2E8 cells had rearranged and expressedκlight-chain genes but displayed them on the surface along with surrogate light chains andμheavy chains. Thus, expression of authentic Tight chain need not coincide with termination of surrogate light-chain utilization in newly formed B cells. Several glycoproteins have recently been demonstrated to be associated with surface immunoglobulin (Ig) on mature B-lineage cells and plasma-cell tumors. We now show that one member of this family (approximately 33 kD) was associated with theμ+surrogate light-chain complex on the 1A9 pre-B-cell clone. When compared to mature B lymphomas, fewer bands coprecipitated with the surface-labeled Ig isolated from pre-B- and early B-cell lines, suggesting that components of the antigen receptor are sequentially acquired during development. The normal replication and differentiation of pre-B cells is probably regulated by complex interactions with multiple cytokines and matrix components of the marrow microenvironment. Cloned lymphocyte lines that are dependent on stromal cells should continue to be important tools for molecular definition of those interactions.

scholarly journals Osteoblast Stimulating Factor-5 Regulates B Lymphopoiesis Via Inhibiting Pre B Cell Proliferation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2926-2926
Author(s):  
Natsuko Fujita ◽  
Kenji Oritani ◽  
Michiko Ichii ◽  
Norimitsu Saitoh ◽  
Kengo Yamawaki ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
B Lymphocyte ◽  
Signal Sequence ◽  
B Lymphopoiesis ◽  
Immature B Cells ◽  
Stimulating Factor

Abstract B lymphopoiesis is a complex and multistep process originated from hematopoietic stem cells (HSC). Recent studies showed that the microenvironment surrounding progenitor cells affects the development of B cells. Soluble factors that bone marrow (BM) stromal cells produce are crucial to the process. For example, SDF1α regulates the survival and homing of HSC and the lineage progenitor cells. IL7 is produced by osteoblasts and promotes the differentiation and proliferation of pre B cells. Although various molecules have been reported for the important roles, there are many proteins, which are secreted from stromal cells and function as a direct regulator of progenitors, remained unknown. In this study, we aimed to find novel secreted or membrane proteins, which modulate B cell differentiation via the microenvironment. We used MS-5 stromal cells to identify the novel secreted regulators because the cell line is known to have the potential to support HSC and B lymphopoiesis. The secreted proteins have signal sequence to pass into endoplasmic reticulum, followed by cell surface expression. Thus, we applied the modified signal sequence trap method (Tashiro et al, Science, 1993). Briefly, the cDNA library from MS-5 was inserted to the HPC4-TF/pEFBOS vector to produce fusion proteins, composed of proteins from cDNA, a HPC4-epitope, and a tissue factor transmembrane. With the screening of each plasmid based on the capacity to express HPC4-epitope on cell surface, we successfully identified 21 secreted or transmembrane proteins, which MS-5 cells produce. Among these proteins, pleiotrophin, proliferin-2 and osteoblast stimulating factor-5 (OSF-5) were selected because of the limited mRNA expression within stromal cell lines. For the functional analysis without the effects on the process of fetus development, we generated transgenic chimera mice (Tg), which produce the indicated protein under the control of kappa chain promoter. As a result, OSF-5, but not pleiotrophin or proliferin-2, Tg showed impaired B lymphocyte development. In OSF-5 chimera mice, the number of B lineage cells was decreased. B220+ cells in the spleen as well as pre B cells and immature B cells in the BM were significantly decreased (B220low CD43low IgM- pre B cells: 2.2 ± 0.2 x 105 cells in OSF-5 Tg vs. 6.7 ± 1.9 x 105 cells in control, B220+ CD43- IgM+ immature B cells: 2.3 ± 0.6 x 105 cells in OSF-5 Tg vs. 5.3 ± 0.8 x 105 cells in control). OSF-5 is widely expressed in mice BM, spleen, thymus, liver, kidney and lung, and the effects on hematopoiesis have never been examined. OSF-5 includes two splicing variants. Variant 1 is a secreted protein and known as aortic carboxypeptidase like protein. Variant 2 is a non-secreted, intracellular protein, known as adipocyte enhancer binding protein. In mice BM, OSF-5 variant 1 is secreted only from stromal cells, while OSF-5 variant 2 is expressed in hematopoietic cells. First, to exclude the cell-intrinsic effects of OSF-5, we ectopically expressed variant 2 in lineage- Sca-1+ c-Kit+ Flt3- HSC and co-cultured them with MS-5. As a result, the generated number of CD19+ B cells was not changed. In contrast, when we knocked down the secreted type of OSF-5 in OP9 stromal cell line to mimic the BM environment, the modified OP9 cells could support the proliferation of pre B cell line, 2E8 more efficiently, compared to control (7.3 ± 1.1 x 105 cells in KD vs. 4.4 ± 0.7 x 105 cells in control). In addition, the knock-down (KD) of variant 1 protein increased the recovered number of CD19+ cells in co-cultures of BM mononuclear cells (6.6 ± 2.8 x 105 cells in KD vs. 4.7 ± 2.8 x 105 cells in control). Finally, we found that colony-forming unit of pre B cells was decreased in the existence of OSF-5 variant 1 (17 ± 8 colonies in variant 1 vs. 198 ± 22 colonies in control). This result indicated that OSF-5 produced by BM stromal cells had a direct effect to inhibit the proliferation of pre B cells. In conclusion, we identified OSF-5 as a BM stromal cell-derived secreted protein, which has an ability to inhibit B lymphopoiesis via regulating the pre B cell proliferation. Our findings could help us to understand molecular regulatory mechanisms of normal B lymphopoiesis as well as causes of B lymphocyte dysregulation, such as change during aging. Disclosures No relevant conflicts of interest to declare.

scholarly journals Establishment of B-Cell Lymphoma Cell Lines Persistently Infected with Hepatitis C Virus In Vivo and In Vitro: the Apoptotic Effects of Virus Infection

2003 ◽  
Vol 77 (3) ◽  
pp. 2134-2146 ◽  
Author(s):  
Vicky M.-H. Sung ◽  
Shigetaka Shimodaira ◽  
Alison L. Doughty ◽  
Gaston R. Picchio ◽  
Huong Can ◽  
...  
Keyword(s):  
Hepatitis C ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Culture System ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Hcv Rna

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Studies of HCV replication and pathogenesis have so far been hampered by the lack of an efficient tissue culture system for propagating HCV in vitro. Although HCV is primarily a hepatotropic virus, an increasing body of evidence suggests that HCV also replicates in extrahepatic tissues in natural infection. In this study, we established a B-cell line (SB) from an HCV-infected non-Hodgkin's B-cell lymphoma. HCV RNA and proteins were detectable by RNase protection assay and immunoblotting. The cell line continuously produces infectious HCV virions in culture. The virus particles produced from the culture had a buoyant density of 1.13 to 1.15 g/ml in sucrose and could infect primary human hepatocytes, peripheral blood mononuclear cells (PBMCs), and an established B-cell line (Raji cells) in vitro. The virus from SB cells belongs to genotype 2b. Single-stranded conformational polymorphism and sequence analysis of the viral RNA quasispecies indicated that the virus present in SB cells most likely originated from the patient's spleen and had an HCV RNA quasispecies pattern distinct from that in the serum. The virus production from the infected primary hepatocytes showed cyclic variations. In addition, we have succeeded in establishing several Epstein-Barr virus-immortalized B-cell lines from PBMCs of HCV-positive patients. Two of these cell lines are positive for HCV RNA as detected by reverse transcriptase PCR and for the nonstructural protein NS3 by immunofluorescence staining. These observations unequivocally establish that HCV infects B cells in vivo and in vitro. HCV-infected cell lines show significantly enhanced apoptosis. These B-cell lines provide a reproducible cell culture system for studying the complete replication cycle and biology of HCV infections.

Insulin release from a cloned precursor beta cell line

1987 ◽  
Vol 113 (1) ◽  
pp. 3-NP ◽  
Author(s):  
K. W. Ng ◽  
P. R. Gummer ◽  
B. L. Grills ◽  
V. P. Michelangeli ◽  
M. E. Dunlop
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Insulin Release ◽  
Cell Lines ◽  
Neonatal Rat ◽  
Insulin Content ◽  
Morphological Evidence ◽  
Bicarbonate Buffer ◽  
Beta Cell Line

ABSTRACT This paper describes the establishment in long-term tissue culture of a functional, clonal beta (B) cell line UMR 407/3 derived from neonatal rat pancreas. Immunofluorescence demonstrated specific and uniform staining for insulin. Transmission electron microscopy showed the presence of microvilli and cytoplasmic granules. The doubling time in culture was approximately 60 h in 2% (v/v) fetal calf serum with inhibition of growth at confluence. Biochemical studies demonstrated the incorporation of [3H]leucine into proinsulin and insulin, with insulin comprising 43·6% of the total radioactivity incorporated into immune complexes. When incubated at 37 °C for 30 min with Krebs–Ringer bicarbonate buffer (pH 7·4), the amount of insulin released on stimulation by 16·7 mmol glucose/l, 20 mmol dl-glyceraldehyde/l or 20 mmol α-ketoisocaproate/l was significantly higher compared with 5·6 mmol glucose/l. The mean insulin content was equivalent to 99±0·4 fmol (s.e.m.)/5 × 105 cells. Regulated insulin release was maintained through at least 15 passages in culture. The cells showed morphological evidence of senescence after passage 26 and this was associated with significant reduction in stimulated insulin release as well as insulin content. The ability of the cells of this clonal line to grow in soft agar suggests that it is a precursor cell line. The clonal B cell lines isolated so far may thus represent variably committed rather than fully differentiated B cells in culture. These clonal non-neoplastic cell lines will be useful models with which to study the regulation of maturation/differentiation of B cells and insulin gene expression. Finally, the method described for the isolation of clonal B cell lines can be used to establish, in culture, precursor clonal lines from other normal tissues. J. Endocr. (1987) 113, 3–10

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