Laurence Guglielmi

The 3′ IgH Locus Control Region Is Sufficient to Deregulate a c-myc Transgene and Promote Mature B Cell Malignancies with a Predominant Burkitt-Like Phenotype

Burkitt lymphoma (BL) features translocations linking c-myc to an Ig locus. Breakpoints in the H chain locus (IgH) stand either close to JH or within switch regions and always link c-myc to the 3′ IgH locus control region (3′ LCR). To test the hypothesis that the 3′ LCR alone was sufficient to deregulate c-myc, we generated mice carrying a 3′ LCR-driven c-myc transgene and specifically up-regulating c-myc in B cells. Splenic B cells from mice proliferated exaggeratedly in response to various signals had an elevated apoptosis rate but normal B220/IgM/IgD expression. Although all Ig levels were lowered in vivo, class switching and Ig secretion proved normal in vitro. Beginning at the age of 12 wk, transgenic mice developed clonal lymphoblastic lymphomas or diffuse anaplastic plasmacytomas with an overall incidence of 80% by 40 wk. Lymphoblastic lymphomas were B220+IgM+IgD+ with the BL “starry sky” appearance. Gene expression profiles revealed broad alterations in the proliferation program and the Ras-p21 pathway. Our study demonstrates that 3′ IgH enhancers alone can deregulate c-myc and initiate the development of BL-like lymphomas. The rapid and constant occurrence of lymphoma in this model makes it valuable for the understanding and the potential therapeutic manipulation of c-myc oncogenicity in vivo.

Platelet-activating Factor and Normal or Leukaemic Haematopoiesis

Platelet-activating factor (PAF), a phospholipid mediator with a wide range of actions on mature leukocytes, acts directly during early human haematopoiesis by affecting the growth of haematopoietic progenitors and indirectly, by modulating cytokine synthesis by bone marrow stromal cells. At this time, its role during leukaemic diseases remains speculative. The lack of membrane PAF receptor (PAF-R) on leukaemic blasts suggest that this receptor represents a marker of mature cells and its membrane induction a consequence of cell maturation. While the couple PAF/PAF-R has been largely studied using B cell lines, few results are available using B cells of patients with haematopoietic malignancies casting some doubts concerning the potential role (if any) of this molecule during leukaemic diseases.

Alterations in plasma angiogenic growth factor concentrations after coronary artery bypass graft surgery: relationships with post-operative complications

To determine whether angiogenic growth factor levels are altered during and after cardiac surgery, plasma concentrations of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and transforming growth factor beta1 (TGFbeta1) were measured in 32 patients undergoing coronary artery bypass graft (CABG) surgery with extracorporeal circulation (ECC). EGF levels significantly decreased during ECC and remained low until the 24th post-operative hour with no difference between complicated and uncomplicated patients. TGFbeta1 and bFGF concentrations significantly increased at the end of ECC and after cross-clamp release, and returned to pre-operative values at the 6th post-operative hour suggesting that the source of these elevations are the lungs and heart. After cross-clamp release bFGF levels but not TGFbeta1 ones were higher in patients with respiratory impairments. VEGF values increased significantly at the 6th and 24th post-operative hours. At the 24th post-operative hour plasma VEGF levels were higher in patients with cardiovascular and hematological impairments. In conclusion, these results highlight that the angiogenic network is profoundly altered in patients undergoing cardiopulmonary bypass as previously demonstrated for lipidic, cytokine and haematopoietic growth factor ones and identify an association between specific post-CABG complications and systemic release of bFGF and VEGF.

The 5′HS4 insulator element is an efficient tool to analyse the transient expression of an Eμ-GFP vector in a transgenic mouse model

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. The 5’HS4 insulator element is an efficient tool to analyse the transient expression of an Eμ-GFP vector in a transgenic mouse model Laurence Guglielmi, Véronique Truffinet, Claire Carrion, Marc Le Bert, Nadine Cogné, Michel Cogné, Yves Denizot

Membrane and Intracellular Platelet-Activating Factor Receptor Expression in Leukemic Blasts of Patients with Acute Myeloid and Lymphoid Leukemia

Platelet‐activating factor (PAF), a phospholipid mediator with a wide range of actions on mature leukocytes, acts through PAF‐receptors (PAF‐Rs) on the membranes of responsive cells. No results are available concerning the putative presence of PAF‐Rs on leukemic blasts. Using multiparameter flow cytometry, we assessed intracellular and membrane PAF‐Rs on blast cells of acute myeloid leukemic (AML) and acute lymphoid leukemic (ALL) patients. Membrane PAF‐Rs were documented in 7/15 cases of ALL and 0/28 cases of AML. Putative intracellular PAF‐Rs were found in blasts of 8/8 ALL and 13/13 AML patients. Vitamin D3 and dimethyl sulfoxide that induced the expression of PAF‐Rs on the membrane of the human promyelocytic leukemia cell line, HL60, failed to induce their expression on the membranes of CD34+ AML blasts. The lack of membrane PAF‐Rs on the membranes of AML blasts confirms that these receptors represent a marker of mature cells and that their membrane induction is a consequence of cell maturation and differentiation.

Detection of functional platelet-activating factor receptors on leukemic B cells of chronic lymphocytic leukemic patients

Although platelet-activating factor receptors (PAF-R) are reported on normal B cells, few results are available concerning leukemic ones. We demonstrated functional PAF-R on cell and nuclear surfaces of leukemic B cells of chronic lymphocytic leukemic (CLL) patients. Analysis of 102 patients revealed dramatic differences for their membrane PAF-R expression, a result that might be related to their plasma IL-4 levels. In the light of the potent immunoregulatory role of PAF on B cell physiology, it is suggested that the presence or absence of PAF-R on leukemic B cells may profoundly affect their in vivo behavior.

Effect of the Eμ IgH enhancer on expression of a GFP reporter gene in transfected B cells and transgenic mice

Transgenic mice were generated to identify the first B cell maturation stage showing expression of an immunoglobulin transcriptional enhancer element (Emu)-green fluorescent protein (GFP) transgene, and to check the ability of the Emu element to behave as a locus control region. Flow cytometry experiments indicated that stably transfected 18-81 cells (a murine pre-B cell line) and A20 cells (a murine IgM(+) B cell line) maintained a constant GFP expression for several months in culture. Contrasting with in vitro results, flow cytometry experiments did not highlight GFP(+) B cells in spleen and bone marrow of Emu-GFP transgenic mice and no GFP transcripts were detected by Northern blot and reverse transcriptase polymerase chain reaction analysis. In transgenic mice, the lack of GFP expression seemed related to transgene DNA methylation occurring within all organs. Our results show dramatic differences for expression of the Emu-GFP transgene in vitro and in vivo. Despite that Emu was reported to efficiently control the in vivo expression of other associated transgenes, it is not sufficient to sustain GFP expression in transgenic mice and to counteract developmental silencing programs that occur in the embryo.

Presence of membrane platelet-activating factor receptors on B cells of chronic B cell leukaemia patients.

Platelet-activating factor (PAF) is an inflammatory phospholipid molecule that acts in vitro on B cell activation, proliferation and immunoglobulin synthesis [1]. PAF acts through membrane PAF receptors (PAF-R) [2]. Recently, we have highlighted their presence on B cells of chronic lymphocytic leukaemia patients [3] and their virtual absence on leukaemic blasts of patients with acute B lymphoid leukaemia [4] suggesting that PAF-R might represent a marker of B cell differentiation and maturation. Taken together, these results lead us to examine for the presence of PAF-R in other mature B cell leukaemias. Blood was obtained from 42 untreated patients according to the Helsinki recommendations. Thirteen patients had mantle B cell lymphoma (6 men, 7 women, mean age 72 years), 15 had a villous or non-villous marginal zone B cell lymphoma (11 men, 4 women, mean age 74 years), 5 had follicular B cell lymphoma (2 men, 3 women, mean age 62 years), 5 had plasma cell leukaemia (2 men, 3 women, mean age 72 years) and 4 had prolymphocytic or prolymphocytoid B cell leukaemia (2 men, 2 women, mean age 77 years). PAF-R were investigated using flow cytometry as previously reported [3,4]. As shown in Fig. 1, B cells of patients with mantle B cell lymphoma, marginal zone B cell lymphoma, follicular lymphoma, prolymphocytic/prolymphocytoid leukaemia and plasma cell leukaemia expressed PAF-R. Using a cut off of 20% PAF-Rþ cells, PAF-R were found in 20% (1/5) of patients with follicular lymphoma, 50% (2/4) of patients with a prolymphocytic/prolymphocytoid leukaemia, 60% (3/5) of patients with a plasma cell leukaemia, 69% (9/13) of patients with a mantle cell lymphoma and 80% (12/15) of patients with a marginal zone B cell lymphoma. Fifty seven (15/26) and 68% (11/16) of patients with a k and a l chain expressed PAF-R, respectively, indicating that the nature of the light chain did not interfere with the presence or absence of membrane PAF-R. This short clinical study highlights PAF-R in several types of chronic (mature) B cell malignancies. It clearly strengthens the hypothesis that the expression of membrane PAF-R is a marker of B cell differentiation and maturation. While the physiological meaning of PAF-R on leukaemic B cells remains an open question, it

No evidence for a putative involvement of platelet-activating factor in systemic lupus erythematosus without active nephritis.

BACKGROUND: Platelet-activating factor (PAF) seems to be implicated in systemic lupus erythematosus (SLE) patients with associated renal diseases. AIMS: In this study, we ensured the role of PAF in SLE patients without renal complications. METHODS: Blood PAF and acetylhydrolase activity, plasma soluble phospholipase A(2), and the presence of antibodies against PAF were investigated in 17 SLE patients without active nephritis and in 17 healthy controls. RESULTS: Blood PAF levels were not different (p=0.45) between SLE patients (6.7+/-2.8 pg/ml) and healthy subjects (9.6+/-3.1 pg/ml). Plasma acetylhydrolase activity (the PAF-degrading enzyme) was significantly (p=0.03) elevated in SLE patients (57.8+/-6.4 nmol/min/ml) as compared with controls (37.9+/-2.6 nmol/min/ml). Plasma soluble phospholipase A(2) (the key enzyme for PAF formation) was not different (p=0.6) between SLE patients (59.1+/-5.1 U/ml) and controls (54.7+/-2.4 U/ml). Antibodies against PAF were detected only in 3/17 SLE patients. Flow cytometry analysis did not highlight PAF receptors on circulating leukocytes of SLE patients. CONCLUSION: This clinical study highlights no evidence for a putative important role of PAF in SLE patients without active nephritis.