ALBERT

Description: Previously, we found that murine bone marrow-derived macrophages (MO) induced in vitro by MO-specific colony-stimulating factor (M-CSF) have little capacity to release prostaglandin E2 (PGE2) and other eicosanoids. This work focused on the functional and transcriptional expression of the key enzymes for the PGE2 synthesis in the MO. Nonadherent bone marrow cells were cultured with RPMI1640 plus 10% fetal bovine serum (FBS) further supplemented with either M-CSF or granulocyte-macrophage (GM)-CSF and interleukin-3 (IL-3). Cellular PGG/H synthase (cyclooxygenase) levels were quantified by cytometric analysis with antibodies specific for the two isozymes of PGG/H synthase (PGG/H synthases 1 and 2). The enzyme activity was monitored by adding exogenous arachidonic acid (AA) substrate to the bone marrow MO cultures and to the cell-free particulate fractions. The levels of PGE2 converted were quantitated by radioimmunoassay (RIA). mRNA levels of the enzymes were detected by Northern blot analysis hybridized with mouse PGG/H synthase cDNA probes, 2.7 kb (PGG/H synthase 1) and 4.2 kb (PGG/H synthase 2). In addition, cellular phospholipase A2 (PLA2) activities were detected with sn-2–14C-arachidonyl phosphatidylcholine as a substrate. Cells proliferating in the presence of GM-CSF and IL-3 for more than 4 days showed significant release of PGE2 (〉 7 ng/10(6) cells) when stimulated by AA. These cells also expressed significant amounts of PGG/H synthase 1 protein, its mRNA (2.7 kb) and cellular PLA2. M-CSF-induced MO, in sharp contrast, expressed little PGG/H synthase protein, mRNA, cellular enzyme activity, or PGE2 release, despite comparable levels of cellular PLA2 activity. These data suggest that the capacity of differentiating marrow-derived MO to form PGE2 is growth factor-dependent.

Description: Previously, we found that murine bone marrow-derived macrophages (MO) induced in vitro by MO-specific colony-stimulating factor (M-CSF) have little capacity to release prostaglandin E2 (PGE2) and other eicosanoids. This work focused on the functional and transcriptional expression of the key enzymes for the PGE2 synthesis in the MO. Nonadherent bone marrow cells were cultured with RPMI1640 plus 10% fetal bovine serum (FBS) further supplemented with either M-CSF or granulocyte-macrophage (GM)-CSF and interleukin-3 (IL-3). Cellular PGG/H synthase (cyclooxygenase) levels were quantified by cytometric analysis with antibodies specific for the two isozymes of PGG/H synthase (PGG/H synthases 1 and 2). The enzyme activity was monitored by adding exogenous arachidonic acid (AA) substrate to the bone marrow MO cultures and to the cell-free particulate fractions. The levels of PGE2 converted were quantitated by radioimmunoassay (RIA). mRNA levels of the enzymes were detected by Northern blot analysis hybridized with mouse PGG/H synthase cDNA probes, 2.7 kb (PGG/H synthase 1) and 4.2 kb (PGG/H synthase 2). In addition, cellular phospholipase A2 (PLA2) activities were detected with sn-2–14C-arachidonyl phosphatidylcholine as a substrate. Cells proliferating in the presence of GM-CSF and IL-3 for more than 4 days showed significant release of PGE2 (〉 7 ng/10(6) cells) when stimulated by AA. These cells also expressed significant amounts of PGG/H synthase 1 protein, its mRNA (2.7 kb) and cellular PLA2. M-CSF-induced MO, in sharp contrast, expressed little PGG/H synthase protein, mRNA, cellular enzyme activity, or PGE2 release, despite comparable levels of cellular PLA2 activity. These data suggest that the capacity of differentiating marrow-derived MO to form PGE2 is growth factor-dependent.

Description: We have previously reported that ex vivo retroviraly-mediated gc gene transfer into CD34 (+) bone marrow precursor cells led to the correction of the immunodeficiency in 9 out of 10 patients with X-linked severe combined immunodeficiency. Follow-up now reaches more that 6 years for the first 2 treated patients. Patients’immune function has been restored. The distribution of both TCR Vb family usage and TCR Vb CDR3 length still reveals a broadly diversified T cell repertoire. Moreover 6 years after treatment the thymus is still seeded by transduced progenitor cells as attested by the presence of TRECS in peripheral blood RTE. Among these patients, three (P4, P5 and P10) developed at 30 to 34 months after gene therapy a monoclonal T cell proliferation requiring a chemotherapy. P4 received also an allogenic HSCT from a MUD but died 26 months after the occurence of the lymphoproliferation. For P5 and P10, chemotherapy has led to an overall control of the clonal proliferation. These two patients are doing well and P5 is off treatment with a good immunological recovery. Genetic analysis of the blastic cells showed that in the two first cases the vector had integrated within or upstream of the LMO2 locus causing an insertional activation of LMO2 transcription. The last case revealed the involvement of several targeted sites, but their exact contribution to the lymphoproliferation is still under investigation. The repeated involvment of LMO2 as a site of vector integration in the proliferating T-cells points to an insertional activation of this gene as at least one of the causes of the oncogenic process. However, the long latency observed in all cases (〉 30 months) suggests that additional “hits” have been required for overt desease. Synergy with gc expression and thereby induced proliferative signals (explaining occurrence in SCID-X1 patients only) is the most obvious hypothesis which we are trying to analyse in a mouse model. A deep analysis of retroviral integration patterns has been performed on patients’PBMCS by LAM-PCR to estimate the frequency of potentially harmful integration events and to assess the risk factors associated with the LTR’s strong enhancer effect of the MLV-based retroviral vector. 708 unique integration sites (IS) have been obtained from all analysed patients post-gene therapy and among them, 577 could be mapped unequivocally to the human genome. *Most of these insertions (63%) are located in the vicinity of 10kb or within the coding sequence of a known gene*. A significant peak of insertion frequency is related closely to the transcription strart site *among the 577 IS, 43 are common integration sites. Among the latter, we found out a high selection of genes involved in human oncogenic process.