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See detailNonmyeloablative stem cell transplantation with CD8-depleted or CD34-selected peripheral blood stem cells.
Baron, Frédéric ULg; Baudoux, Etienne ULg; Frere, Pascale ULg et al

in Journal of Hematotherapy & Stem Cell Research (2002), 11(2), 301-14

To decrease the incidence of graft-versus-host disease (GVHD) observed after nonmyeloablative stem cell transplantation (NMSCT), we studied the feasibility of CD8-depleted or CD34-selected NMSCT followed ... [more ▼]

To decrease the incidence of graft-versus-host disease (GVHD) observed after nonmyeloablative stem cell transplantation (NMSCT), we studied the feasibility of CD8-depleted or CD34-selected NMSCT followed by CD8-depleted preemptive donor lymphocyte infusion (DLI) given in incremental doses on days 40 and 80. Fourteen patients with high-risk malignancies and an HLA-identical sibling (n = 8) or alternative donor (n = 6) but ineligible for a conventional transplant were included. Nonmyeloablative conditioning regimen consisted in 2 Gy total body irradiation (TBI) alone, 2 Gy TBI and fludarabine (previously untreated patients) or cyclophosphamide and fludarabine (patients who had previously received > or =12 Gy TBI). Patients 1-4 (controls) received unmanipulated peripheral blood stem cells (PBSC) and DLI and patients 5-14 CD8-depleted or CD34-selected PBSC followed by CD8-depleted DLI. Post-transplant immunosuppression was carried out with cyclosporine A (CsA) and mycophenolate mofetil (MMF). Initial engraftment was seen in all patients, but 1 patient (7%) later rejected her graft. The actuarial 180-day incidence of grades II-IV acute GVHD was 75% for patients 1-4 versus 0% for patients 5-14 (p = 0.0019). Five of 14 patients were in complete remission (CR) 180 days after the transplant and 6/14 had partial responses. The 1-year survival rate was 69%, and nonrelapse and relapse mortality rates were 16 and 18%, respectively. We conclude that CD8-depleted or CD34-selected NMSCT followed by CD8-depleted DLI is feasible and considerably decreases the incidence of acute GVHD while preserving engraftment and apparently also the graft-versus-leukemia (GVL) effect. Further studies are needed to confirm this encouraging preliminary report. [less ▲]

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See detailIncreased binding and defective migration across fibronectin of cycling hematopoietic progenitor cells.
Giet, Olivier ULg; Van Bockstaele, Dirk R; Di Stefano, Ivano et al

in Blood (2002), 99(6), 2023-31

Engraftment of hematopoietic progenitor cells has been shown to decrease during cell cycle transit. We studied cell cycle-associated changes in adhesion and migration of mitotically activated cord blood ... [more ▼]

Engraftment of hematopoietic progenitor cells has been shown to decrease during cell cycle transit. We studied cell cycle-associated changes in adhesion and migration of mitotically activated cord blood CD34+ cells. Migration toward medium conditioned by the stromal-derived factor-1-producing cell line MS-5 was studied in bovine serum albumin- and fibronectin (Fn)-coated transwells. Migration was reduced in cycling CD34+ cells and long-term culture-initiating cells (LTC-ICs) compared with their noncycling counterparts across Fn but not across bovine serum albumin. Conversely, Fn binding was higher in cycling CD34+ cells and LTC-ICs compared with noncycling progenitor cells, while adhesion of both subsets to bovine serum albumin was undetectable. The contribution of alpha4 and alpha5 integrins in mediating adhesion and migration of activated CD34+ cells onto Fn was analyzed by neutralization experiments. While alpha4-mediated Fn binding decreased during G(2)/M, alpha5 integrin-mediated adhesion increased during transit from G(0)/G(1) to S and G(2)/M phases. As for migration, the contribution of alpha4 integrin was similar in all phases, whereas alpha5-directed migration was lower in G(2)/M compared with G(0)/G(1) and S phases. Defective migration of cycling CD34+ cells was not due to differences in alpha5 integrin expression. In conclusion, chemotaxis across Fn is less efficient in cycling progenitor cells in correlation with an increased Fn binding capacity. In addition, alpha4 and alpha5 integrin functions are independently modulated during cell cycle transit. [less ▲]

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See detailInvolvement of Insulin-Like Growth Factors in Early T Cell Development: A Study Using Fetal Thymic Organ Cultures
Kecha, O.; Brilot, F.; Martens, Henri ULg et al

in Endocrinology (2000), 141(3), 1209-17

The expression of insulin-like growth factor (IGF) and IGF receptor genes was investigated by RT-PCR during ontogeny of the murine thymus. IGF-1, IGF-1R, M6P/IGF-2R genes are expressed in the thymus both ... [more ▼]

The expression of insulin-like growth factor (IGF) and IGF receptor genes was investigated by RT-PCR during ontogeny of the murine thymus. IGF-1, IGF-1R, M6P/IGF-2R genes are expressed in the thymus both in fetal and postnatal life, whereas IGF-2 messenger RNAs (mRNAs) decline after birth but are still detectable on the seventh week. By in situ hybridization, IGF-2 transcripts were located in the outer cortex and medulla of the postnatal thymus, and on the whole surface ofthe epithelial-like network in the fetal thymus. The effects of anti-IGFs and IGF-receptors neutralizing Abs on the generation of pre-T cell subpopulations were then investigated using fetal thymic organ cultures (FTOC). FTOC treatment with an anti-IGF-2 mAb, an anti-IGF-1R mAb, or an anti-M6P/IGF-2R polyclonal Ab induced a blockade of T cell differentiation at the CD4-CD8- stage, as shown by a significant increase in the percentage of CD4-CD8- cells and a decrease in the percentage of CD4+CD8+ cells. Moreover, anti-IGF-2 Ab treatment induced an increase in CD8+ cells suggesting that thymic IGF-2 might have a role in determining differentiation into the CD4 or CD8 lineage. Anti-IGF-1 Ab treatment decreased the proportion in CD4-CD8- cells and increased the frequency in CD4+CD8+. FTOC treatment with anti-(pro)insulin did not exert any significant effect on T cell development. These data indicate that the intrathymic IGF-mediated signaling plays an active role in the early steps of T cell differentiation during fetal development. [less ▲]

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See detailReactive Oxygen Intermediate-Dependent Nf-Kappab Activation by Interleukin-1beta Requires 5-Lipoxygenase or Nadph Oxidase Activity
Bonizzi, Giuseppina; Piette, Jacques ULg; Haterte, Stéphanie ULg et al

in Molecular & Cellular Biology (1999), 19(3), 1950-60

We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-kappaB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are ... [more ▼]

We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-kappaB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are yet to be determined and might include 5-lipoxygenase (5-LOX) and NADPH oxidase. 5-LOX and 5-LOX activating protein (FLAP) are coexpressed in lymphoid cells but not in monocytic or epithelial cells. Stimulation of lymphoid cells with interleukin-1beta (IL-1beta) led to ROI production and NF-kappaB activation, which could both be blocked by antioxidants or FLAP inhibitors, confirming that 5-LOX was the source of ROIs and was required for NF-kappaB activation in these cells. IL-1beta stimulation of epithelial cells did not generate any ROIs and NF-kappaB induction was not influenced by 5-LOX inhibitors. However, reintroduction of a functional 5-LOX system in these cells allowed ROI production and 5-LOX-dependent NF-kappaB activation. In monocytic cells, IL-1beta treatment led to a production of ROIs which is independent of the 5-LOX enzyme but requires the NADPH oxidase activity. This pathway involves the Rac1 and Cdc42 GTPases, two enzymes which are not required for NF-kappaB activation by IL-1beta in epithelial cells. In conclusion, three different cell-specific pathways lead to NF-kappaB activation by IL-1beta: a pathway dependent on ROI production by 5-LOX in lymphoid cells, an ROI- and 5-LOX-independent pathway in epithelial cells, and a pathway requiring ROI production by NADPH oxidase in monocytic cells. [less ▲]

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See detailFlow cytometry estimation of nuclear size and ploidy level of habituated calli of sugar beet
Kevers, Claire ULg; Greimers, Roland ULg; Franck, Thierry ULg et al

in Biologia Plantarum (1999), 42(3), 321-332

A fully habituated (auxin- and cytokinin-independent) self-regenerating (organogenic) sugar beet cell line (HO) and a fully habituated non-organogenic one (HNO) derived from the former one, were analyzed ... [more ▼]

A fully habituated (auxin- and cytokinin-independent) self-regenerating (organogenic) sugar beet cell line (HO) and a fully habituated non-organogenic one (HNO) derived from the former one, were analyzed as to their nuclear size and DNA content. Flow cytometry and image analysis were used and cells of certified diploid leaves of the same sugar beet strain served as controls. The HNO cells had been shown previously to have many characteristics of cancerous cells. The analyses made on leaves and HNO cells indicated the presence of only one population of cycling cells. In HO cells. two cycling populations were detected: the first one had the same DNA content as the leaves while the second one contained two fold more DNA than the first population. HNO cells showed the higher nuclear size and DNA content. HNO cells also showed evidence of aneuploidy. Thus, nuclear size, DNA content and ploidy level increase together with the neoplasic progression to culminate in HNO cells with the loss of organogenic totipotency. [less ▲]

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See detailThymic insulin-related polypeptides: role in T cell selection and in central self-tolerance of the insulin family
Kecha, Ouafae; Brilot, Fabienne; Martens, Henri ULg et al

in The Endocrine Society (Ed.) Proceedings of the 81st Annual Meeting of the Endocrine Society (1999)

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See detailIn Vitro Propagated Dendritic Cells from Patients with Human-Papilloma Virus-Associated Preneoplastic Lesions of the Uterine Cervix: Use of Flt3 Ligand
Hubert, Pascale ULg; Greimers, Roland ULg; Franzen-Detrooz, E. et al

in Cancer Immunology, Immunotherapy (1998), 47(2), 81-9

Dendritic cells (DC) are the most efficient antigen presenting cells. The clinical use of DC as vectors for antitumor and anti-infectious disease immunotherapy has been limited by their low level and ... [more ▼]

Dendritic cells (DC) are the most efficient antigen presenting cells. The clinical use of DC as vectors for antitumor and anti-infectious disease immunotherapy has been limited by their low level and accessibility in normal tissue. Substantial numbers of DC can be generated from peripheral blood cultured in the presence of interleukin-4 (IL-4) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). We showed in this study that substantial numbers of DC can be obtained from the peripheral blood of patients with (pre)neoplastic lesions of the uterine cervix. The procedure required relatively small blood samples (10 ml) and the presence of 100 U/ml IL-4 and 800 U/ml GM-CSF in the culture medium. There was no significant difference in the morphology, yield, phenotype and function of generated DC between patients with cervical (pre)neoplastic lesions and healthy individuals. When the hematopoietic factor Flt3 ligand (Flt3L, 40 ng;ml) was added, there was an average increase in the DC population of 26% compared to cultures with GM-CSF and IL-4 alone. Approximately 1.2 x 10(6) cells with the characteristics of dendritic cells could be obtained when Flt3L was included in the medium. The addition of Flt3L did not modify the phenotypic profile of DC (HLA-DR+, CD1a+, CD4+, CD54+, CD80+, CD86+. CD40+, CD3- and CD14-). In addition, Flt3L generated functional DC capable of stimulating the proliferation of alloreactive T cells. These results suggest that Flt3L, in association with GM-CSF and IL-4, provides an advantageous tool for the large-scale generation of DC and that an immunotherapy based on the use of DC generated in vitro is possible in patients with (pre)neoplastic lesions of the uterine cervix. [less ▲]

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See detailCd28-B7 Costimulatory Blockade by Ctla4ig Delays the Development of Retrovirus-Induced Murine Aids
de Leval, Laurence ULg; Colombi, S.; Debrus, S. et al

in Journal of Virology (1998), 72(6), 5285-90

Mouse AIDS (MAIDS) induced in C57BL/6 mice by infection with a replication-defective retrovirus (Du5H) combines extensive lymphoproliferation and profound immunodeficiency. Although B cells are the main ... [more ▼]

Mouse AIDS (MAIDS) induced in C57BL/6 mice by infection with a replication-defective retrovirus (Du5H) combines extensive lymphoproliferation and profound immunodeficiency. Although B cells are the main target of viral infection, recent research has focused on CD4(+) T cells, the activation of which is a key event in MAIDS induction and progression. A preliminary observation of increased expression of B7 molecules on B cells in MAIDS prompted us to address the possible involvement of the CD28/B7 costimulatory pathway in MAIDS. Mice infected with the MAIDS-inducing viral preparation were treated with murine fusion protein CTLA4Ig (3 x 50 microg/week given intraperitoneally), a competitive inhibitor of physiological CD28-B7 interactions. In CTLA4Ig-treated animals, the onset of the disease was delayed, lymphoproliferation progressed at a much slower rate than in untreated mice, and the loss of in vitro responsiveness to mitogens was reduced. Relative expression of Du5H did not differ between treated and untreated animals. These results suggest that the CD28/B7 costimulatory pathway contributes to MAIDS development. [less ▲]

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See detailThe DNA intercalating alkaloid cryptolepine interferes with topoisomerase II and inhibits primarily DNA synthesis in B16 melanoma cells.
Bonjean, K.; De Pauw, Marie-Claire ULg; Defresne, Marie-Paule ULg et al

in Biochemistry (1998), 37(15), 5136-46

Cryptolepine hydrochloride is an indoloquinoline alkaloid isolated from the roots of Cryptolepis sanguinolenta. It is characterized by a multiplicity of host-mediated biological activities, including ... [more ▼]

Cryptolepine hydrochloride is an indoloquinoline alkaloid isolated from the roots of Cryptolepis sanguinolenta. It is characterized by a multiplicity of host-mediated biological activities, including antibacterial, antiviral, and antimalarial properties. To date, the molecular basis for its diverse biological effects remains largely uncertain. Several lines of evidence strongly suggest that DNA might correspond to its principal cellular target. Consequently, we studied the strength and mode of binding to DNA of cryptolepine by means of absorption, fluorescence, circular, and linear dichroism, as well as by a relaxation assay using DNA topoisomerases. The results of various optical and gel electrophoresis techniques converge to reveal that the alkaloid binds tightly to DNA and behaves as a typical intercalating agent. In DNAase I footprinting experiments it was found that the drug interacts preferentially with GC-rich sequences and discriminates against homo-oligomeric runs of A and T. This study has also led to the discovery that cryptolepine is a potent topoisomerase II inhibitor and a promising antitumor agent. It stabilizes topoisomerase II-DNA covalent complexes and stimulates the cutting of DNA at a subset of preexisting topoisomerase II cleavage sites. Taking advantage of the fluorescence of the indoloquinoline chromophore, fluorescence microscopy was used to map cellular uptake of the drug. Cryptolepine easily crosses the cell membranes and accumulates selectively into the nuclei rather than in the cytoplasm of B16 melanoma cells. Quantitative analyses of DNA in cells after Feulgen reaction and image cytometry reveal that the drug blocks the cell cycle in G2/M phases. It is also shown that the alkaloid is more potent at inhibiting DNA synthesis rather than RNA and protein synthesis. Altogether, the results provide direct evidence that DNA is the primary target of cryptolepine and suggest that this alkaloid is a valid candidate for the development of tumor active compounds. [less ▲]

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See detailRegulation of major histocompatibility complex class I expression by NF-kB-related proteins in breast cancer cells
Dejardin, Emmanuel ULg; Deregowski, Valérie; GREIMERS, Roland ULg et al

in Oncogene (1998)

Downregulation of MHC Class I antigens has been observed in many cancers and usually results from a decreased gene transcription. A reporter CAT gene dependent on the MHC Class I kB site or on a longer ... [more ▼]

Downregulation of MHC Class I antigens has been observed in many cancers and usually results from a decreased gene transcription. A reporter CAT gene dependent on the MHC Class I kB site or on a longer promoter is transactivated by NF-kB complexes contain- ing p65 or RelB. p100 as well as IkB-a are potent inhibitors of this transcription and p100 sequesters RelB and p65 complexes in the cytoplasm of breast cancer cells. However, although p100 is highly expressed in a number of breast cancer cell lines, MHC Class I antigen expression was observed on all the cell lines we analysed and could be further induced by stimulation with the cytokines IFN-g or TNF-a. Stable transfection of a unresponsive mutated IkB-a Ser 32-36 expression vector showed that TNF-a induced MHC Cl I expression in an NF-kB-dependent way while IFN-g did it independently of any NF-kB activation. [less ▲]

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See detailReciprocal regulation of protein tyrosine kinases p56lck and p59fyn, and altered tyrosine phosphorylation in murine AIDS.
Trebak, Mohamed; Lambert, Chantal ULg; Rahmouni, Souad ULg et al

in International Immunology (1998), 10(10), 1473-80

Murine AIDS (MAIDS), caused by a defective murine leukemia virus, is a severe lymphoproliferative disease associated with profound immunodeficiency and increased susceptibility to opportunistic infections ... [more ▼]

Murine AIDS (MAIDS), caused by a defective murine leukemia virus, is a severe lymphoproliferative disease associated with profound immunodeficiency and increased susceptibility to opportunistic infections. Most subsets of lymphocytes, including CD4+ and CD8+ T cells, are refractory to mitogen stimulation. As a first step to examine proximal signal transduction in the infected mice, Western and Northern blot analyses were performed, and showed that p56lck is dramatically decreased at the protein as well as the mRNA level in the lymph nodes (LN). In contrast, p59(fyn) and its mRNA were slightly increased in the LN of the same mice. Similar results were obtained with purified T cells. Interestingly, the thymus of the infected animals did not show any abnormality regarding p56(lck) or p59(fyn). Tyrosine phosphorylation was constitutively increased in the infected mice and was barely amplified by anti-CD3 mAb stimulation. A similar pattern was observed when tyrosine phosphorylation was selectively examined at the level of ZAP-70. Our results suggest that a reciprocal regulation of p56(lck) and p59(fyn) protein tyrosine kinases, previously described in various models of anergy, could also be involved in the pathogenesis of MAIDS. [less ▲]

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See detailApoptosis During the Development of Radiogenic Thymic Lymphomas: Effects of Treatments Inhibiting Lymphoma Development
Humblet, Chantal ULg; Denis, Ghislaine; Greimers, Roland ULg et al

in Anticancer Research (1998), 18(5A, Sep-Oct), 3469-74

INTRODUCTION: Whole body fractionated irradiation induces thymic lymphomas in C57BL/Ka mice after a latent period during which preleukemic cells progressively transform into leukemic cells within an ... [more ▼]

INTRODUCTION: Whole body fractionated irradiation induces thymic lymphomas in C57BL/Ka mice after a latent period during which preleukemic cells progressively transform into leukemic cells within an abnormal thymic microenvironment. A bone marrow graft or repeated cytokine injections prevent lymphoma development. We think that these treatments restore altered mechanisms controlling apoptosis. MATERIALS AND METHODS: Apoptosis was analyzed by flow cytometry in thymocytes from different groups of mice (control, preleukemic, prevented mice). RESULTS: The apoptotic rates did not change in freshly isolated thymocytes from different experimental groups. However, after culture, the level of apoptosis increased in preleukemic thymuses; and returned to normal value in cultured thymocytes from irradiated mice after lymphoma preventing treatments. Furthermore, thymic microenvironmental factors can control thymocyte apoptosis. CONCLUSION: We propose that after leukemogenic irradiation, there is an increase of cells with an activated suicide program, but that alterations of thymic environmental factors rescue them from apoptosis, allowing their further neoplastic transformation. [less ▲]

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See detailSubset-Specific Analysis of Calcium Fluxes in Murine Aids
Moutschen, Michel ULg; Trebak, M.; Greimers, Roland ULg et al

in International Immunology (1996), 8(11), 1715-27

Infection of susceptible strains of mice with the Duplan strain of murine leukemia viruses induces a syndrome called MAIDS (murine acquired immunodeficiency syndrome) characterized by immunodeficiency and ... [more ▼]

Infection of susceptible strains of mice with the Duplan strain of murine leukemia viruses induces a syndrome called MAIDS (murine acquired immunodeficiency syndrome) characterized by immunodeficiency and lymphoproliferation. In addition to a complete refractoriness of most subsets of lymphocytes to mitogen stimulation, the development of phenotypic abnormalities occurs such as the appearance of an abnormal CD4+ T cell subset lacking membranes Thy-1. This study was performed to compare the calcium responses during the early stages of MAIDS (week 9 or earlier) between T cells and B cells and between CD4+Thy-1- and CD4+Thy-1+ T cells. B cells were strikingly less affected than T cells: their baseline [Ca2+]i did not significantly increase, and their calcium response to anti-IgM antibody and concanavalin A (Con A) was partially maintained. In contrast, the response to Con A was completely abolished in T cells. Interestingly, calcium mobilization in response to membrane receptor-independent stimuli such as ionophores and thapsigargin was strongly inhibited in T cells, while no such inhibition was found in B cells. In comparison with their CD4+Thy-1+ counterparts, CD4+Thy-1- T cells had blunted calcium responses in controls, as well as in infected mice. However, CD4+Thy-1+ T cells were also strikingly altered, suggesting that the loss of membrane Thy-1 could be associated with, but not directly responsible for abnormalities of calcium responses in CD4+ T cells from RadLV-Rs-infected mice. [less ▲]

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See detailFurther Characterization of Cytotoxic T Cells Generated by Short-Term Culture of Human Peripheral Blood Lymphocytes with Interleukin-2 and Anti-Cd3 Mab
Jacobs, Nathalie ULg; Greimers, Roland ULg; Mazzoni, Alexandra et al

in Cancer Immunology, Immunotherapy (1996), 42(6), 369-75

In this study we have specifically investigated the participation of T cells in the cytotoxic activity of peripheral blood lymphocytes (PBL) activated by interleukin-2 (IL-2, 50 U/ml) alone or in ... [more ▼]

In this study we have specifically investigated the participation of T cells in the cytotoxic activity of peripheral blood lymphocytes (PBL) activated by interleukin-2 (IL-2, 50 U/ml) alone or in combination with an anti-CD3 mAb (BMA030, 10 ng/ml, IgG2a). Purified CD3+ T cells, incubated in the presence of the anti-CD3 mAb for 4 days, mediated a cytotoxic activity against HL60 and U937 tumor cell lines. Several findings suggested the involvement of a redirected-cytotoxicity phenomenon, since the lytic process was restricted to target cell lines bearing the high-affinity Fc gamma receptor (Fc gamma RI) and T lymphocytes stimulated by IL-2 alone did not lyse these cell lines. Furthermore, anti-CD3 mAb F(ab')2, anti-CD3 IgG1 (UCHT1), phytohemagglutinin or staphylococcal enterotoxin A did not induce a similar cytotoxic activity in T lymphocytes. The cytotoxic process occurred in the presence of a very low level of anti-CD3 antibodies (in the nanomolar range). The cytotoxic activity of T cells stimulated by IL-2 or by IL-2 + BMA030, against OVCAR-3 cells (MOv18+ ovarian tumor cell line), was also compared in the presence of a bispecific antibody OC/TR, anti-CD3 x MOv18). The stimulation by IL-2 + BMA030 induced approximately a twofold higher cytotoxic activity than IL-2-activated T cells. This could be related to the state of activation of effector cells stimulated by IL-2 + BMA030, since the phenotypic analysis showed an increased proportion of T cells expressing several activation/differentiation markers (CD25, HLA-DR, CD45R0, adhesion molecules). These findings could be applied to the design of therapeutic protocols using anti-CD3 x antitumoral bispecific antibodies. [less ▲]

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See detailPrevention of Murine Radiogenic Thymic Lymphomas by Tumor Necrosis Factor or by Marrow Grafting
Humblet, Chantal ULg; Greimers, Roland ULg; Delvenne, Philippe ULg et al

in Journal of the National Cancer Institute (1996), 88(12), 824-31

BACKGROUND: Split-dose irradiation (1.75 Gy given weekly for 4 weeks) of C57BL/Ka mice induces the emergence of preleukemic cells (PLCs). These cells develop into leukemic cells after a latency period of ... [more ▼]

BACKGROUND: Split-dose irradiation (1.75 Gy given weekly for 4 weeks) of C57BL/Ka mice induces the emergence of preleukemic cells (PLCs). These cells develop into leukemic cells after a latency period of 3-6 months. The survival and transformation of PLCs are dependent on radiation-induced alterations of the thymic epithelium and of resident lymphocyte (i.e., thymocyte) subpopulations in the thymus. PLCs can be eliminated, concomitantly with the restoration of the thymus, by grafting bone marrow cells immediately after the last irradiation. Our hypothesis was that any agent able to restore the thymus after leukemogenic irradiation would exert the same effects as a bone marrow graft. Tumor necrosis factor-alpha (TNF-alpha) is one such possible agent, since it has been shown to modulate some functions of the thymic epithelium and thymocyte subpopulations. PURPOSE: The goal of this study was to assess the ability of repeated intraperitoneal injections of TNF-alpha to functionally replace bone marrow transplantation in the restoration of normal intrathymic lymphopoiesis and in the prevention of thymic lymphomas in split-dose-irradiated mice. METHODS: We replaced the bone marrow graft with repeated injections of TNF-alpha (25 000 U/injection) in the split-dose-irradiated (4 x 1.75 Gy) C57BL/Ka mouse model. We analyzed the expression of the cell differentiation markers CD4 and CD8 on thymocytes by flow cytometry. We also studied the thymic environment by isolating thymic nurse cells, the bone marrow prothymocyte activity by analyzing thymic repopulation, and the evolution of PLCs by an in vivo transplantation assay. Local production of TNF-alpha after bone marrow grafting was examined by in situ hybridization. Injections of anti-TNF-alpha antibodies were given to split-dose-irradiated mice to test the effect of neutralizing TNF-alpha in vivo. One-way analysis of variance and Newman-Keuls two-tailed tests were used to test statistical significance. RESULTS: Multiple injections of TNF-alpha into split-dose-irradiated mice did not influence bone marrow prothymocyte activity but restored thymocyte subpopulations and thymic epithelium, induced the disappearance of PLCs, and prevented the development of lymphomas. Moreover, a bone marrow graft significantly stimulated intrathymic production of TNF-alpha messenger RNA (P<.01), and anti-TNF-alpha antibodies partially inhibited the antilymphomatous effects of bone marrow graft in split-dose-irradiated mice (P<.05). CONCLUSION: These data strongly suggest that TNF-alpha is a mediator that is involved in the mechanisms by which bone marrow transplantation functions to prevent thymic lymphomas in split-dose-irradiated mice. IMPLICATIONS: Cytokines might be used in some biological systems, particularly in the hemopoietic system, as a therapeutic agent for the secondary prevention of cancer. [less ▲]

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See detailImproved Four-Color Flow Cytometry Method Using Fluo-3 and Triple Immunofluorescence for Analysis of Intracellular Calcium Ion ([Ca2+]I) Fluxes among Mouse Lymph Node B- and T-Lymphocyte Subsets
Greimers, Roland ULg; Trebak, M.; Moutschen, Michel ULg et al

in Cytometry (1996), 23(3), 205-17

A visible-light, dual-laser, flow cytometric method was developed for the simultaneous analysis of intracellular ionized calcium concentration ([Ca2+]i) and three cell-surface markers (CD4, CD8, and Thy-1 ... [more ▼]

A visible-light, dual-laser, flow cytometric method was developed for the simultaneous analysis of intracellular ionized calcium concentration ([Ca2+]i) and three cell-surface markers (CD4, CD8, and Thy-1.2 antigens) by using the calcium probe fluo-3 and using R-phycoerythrin (PE), peridinin chlorophyll-alpha protein (PerCP), and allophycocyanin (APC) conjugated monoclonal antibodies (MoAbs). This improved method was used in the analysis of [Ca2+]i mobilization upon in vitro stimulation with mitogenic lectins [phytohaemagglutinin (PHA) or concanavalin A (ConA)], anti-CD3 MoAbs, or A23187 calcium ionophore in the heterogeneous lymph node cell populations from healthy C57BL/Ka mice. The present results show that the calcium responses were heterogeneous and dependent on the cellular immunophenotype, not only on lectins or anti-CD3 MoAbs stimulation, but also on the receptor-independent A23187 ionophore stimulation. An in situ fluo-3 calibration method (using A23187 and metabolic poisons in Ca2+ /EGTA buffers with known free calcium concentrations) indicated a resting [Ca2+]i in lymphocytes of 103 +/- 23 nM (mean +/- S.D.) but with significant differences between the [Ca2+]i in B cells and in all of the T-cell subsets (CD4+Thy-1+, CD4+Thy-1-, and CD8+T cells). Both the B cells and the T-cell subsets showed an increase of fluo-3 fluorescence upon in vitro stimulation with ConA or PHA, but the calcium mobilization following lectin stimulation was time delayed in all T-cell subsets. Only the T cells, including the CD4+Thy-1- subset, responded to anti-CD3 MoAbs. The percentage of responding cells upon stimulation with ConA was higher in T cells than in B cells. By contrast, PHA gave a higher response in B cells. After stimulation with different mitogens, [Ca2+]i increased in both CD4+ and CD8+ T-cell subsets. However, the percentage of responding cells was far higher in the CD4+Thy-1+ subset than in the CD4+Thy-1- or the CD8+T-cell subsets. The stimulation with A23187 ionophore induced a higher calcium response in B cells than in T cells. Interestingly, it also induced greater Ca2+ mobilization in CD4+ than in CD8+T cells. These results demonstrate the potential use of fluo-3 simultaneously with three fluorescein (FITC)-compatible fluorochromes. This technique may be useful for investigating the role of the CD4+Thy-1-T cells, a rare subset that is abnormally expanded in a murine acquired immunodeficiency syndrome (murine AIDS). [less ▲]

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See detailSpontaneous and Induced Apoptosis after Whole Body Radiation Exposure: Experimental Approaches. Observations in Radio-Induced Thymic Lymphomagenesis
Humblet, Chantal ULg; Deman, J.; Franzen, Rachelle ULg et al

in Stem Cells (1995), 13(Suppl 1), 129-35

Radio-induced thymic lymphomagenesis is associated with alterations in the balance between thymocyte subsets and cytokinetic perturbations. The objectives of this work were to investigate whether these ... [more ▼]

Radio-induced thymic lymphomagenesis is associated with alterations in the balance between thymocyte subsets and cytokinetic perturbations. The objectives of this work were to investigate whether these alterations are associated with alterations in the basic levels of thymocyte apoptosis. For this purpose, we tested DNA fragmentation by gel electrophoresis, analyzed DNA content by propidium iodide staining of ethanol fixed cells and looked for DNA strand breaks on tissue sections by in situ end labeling. We described an increase of the levels of apoptosis in cultured thymocytes during the preleukemic period, while the basic levels of apoptosis observed in situ are similar in normal and in preleukemic thymuses. We propose that after leukemogenic irradiations, there is an increase of cells wherein the cell suicide program is activated but that environmental thymic factors rescue them from apoptosis. Preleukemic cells could belong to this abnormally surviving population of cells "programmed to die," wherein additional genomic abnormalities would lead to fully neoplastic transformation. [less ▲]

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See detailAgm-1470, a Potent Angiogenesis Inhibitor, Prevents the Entry of Normal but Not Transformed Endothelial Cells into the G1 Phase of the Cell Cycle
Antoine, Nadine ULg; Greimers, Roland ULg; De Roanne, C. et al

in Cancer Research (1994), 54(8), 2073-6

AGM-1470 is a potent angiogenesis inhibitor that is very effective in inhibiting endothelial cell proliferation in both in vitro and in vivo models and that prevents tumor growth in vivo. Although this ... [more ▼]

AGM-1470 is a potent angiogenesis inhibitor that is very effective in inhibiting endothelial cell proliferation in both in vitro and in vivo models and that prevents tumor growth in vivo. Although this molecule appears to be a most promising anticancer drug, its mechanism of action has not yet been elucidated. In this study, we examined the effects of AGM-1470 on the cell cycle of normal and transformed endothelial cells. We showed that AGM-1470, at picomolar concentrations, specifically inhibits the proliferation of both bovine aortic endothelial cells and human umbilical vein endothelial cells. AGM-1470 was ineffective in significantly inhibiting the proliferation of Ea.hy926 cells, a hybrid cell line obtained by the fusion of human umbilical vein endothelial cells with a human carcinoma cell line, or cEnd.1 cells, a polyoma middle T oncogene-transformed endothelioma cell line derived from mouse embryo. Using a double labeling technique with anti-Ki67 antibodies and propidium iodide, we demonstrated, with flow cytometry analysis, that AGM-1470 specifically prevents the entry of endothelial cells into the G1 phase of the cell cycle. We also showed that AGM-1470 was ineffective in inhibiting endothelial cell migration toward laminin or capillary-like tube formation inside a type I collagen matrix induced by phorbol esters. Our data strongly suggest that AGM-1470 is a molecule that specifically inhibits a cell cycle control pathway active in normal cells but which could be bypassed or altered in transformed cells. [less ▲]

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