In the adult liver, the plasma membrane Liver Regulating Protein (LRP) has been found to participate in the recognition signals between hepatocytes and rat liver epithelial cells (RLECs) and to play a critical role in the functional stability of hepatocytes. Here, we report the involvement of LRP in hematopoiesis. First, LRP was evidenced in the hematopoietic fetal rat liver and in adult rat bone marrow. Then, the involvement of LRP in adult rat and human hematopoiesis was investigated in coculture of hematopoietic cells with RLECs. In the rat, RLECs sustain long-term production of hematopoietic cells and committed progenitors as well as bone marrow stroma cells. These effects can be specifically blocked by addition of anti-LRP antibodies to both culture systems. The supportive activity of RLECs on human hematopoiesis was assessed by comparison to the reference MS-5 cell line from both unifractioned and purified CD34(+)CD38(-) hematopoietic cells. Taken together, our results argue for the involvement of LRP in heterotypic interaction signals mediated by RLEC and bone marrow stromal cells which lead to hematopoietic primitive progenitors development. They might lead to interesting applications in fundamental research, pharmacology and therapeutics.

61 autologous transplantations for haematological malignancies have been performed with peripheral blood stem cells in 60 patients. 26 grafts were performed for non-Hodgkin's lymphoma (18 patients were transplanted in first remission, 8 patients after progression or relapse), 13 for multiple myeloma, 7 for Hodgkin's disease and 10 for acute myeloid leukaemia. One patient died from thrombosis of the portal vein. 38 patients were in complete remission 29 months (extremes: 14-44) after transplantation. 21 of 60 patients progressed or relapsed after transplantation, and 14 died. No death was attributed to graft failure, infection or haemorrhage. In conclusion, transplantation with peripheral blood stem cells is well tolerated, has a low toxicity rate, and can be used safely for patients with haematological malignancies.

119 collections of peripheral blood stem cells were performed in 60 patients suffering from various haematological malignancies. One patient was transplanted twice. Peripheral blood stem cell mobilisation was performed using various regimens. A correlation (r = 0.732) was found between the number of peripheral CD34+ cells and the number of CD34+ cells that were collected by apheresis. Furthermore, the number of collected peripheral blood stem cells correlated with the number of colony-forming units (CFU-GM) identified after cell culture (r = 0.607). After transplantation, the duration of neutropenia (> 0.5 x 10(9)/l) was 11.7 days (SD = 3.3) and of thrombocytopenia (> 20 x 10(9)/l) 12.7 days (SD = 6.8). For the whole group, no correlation was observed between the number of CD34+ cells infused and the length of the aplasia. However, when the patients were separated into two groups, according to the amount of CD34+ cells transplanted (< 10 x 10(6)/kg, n = 48 and > 10 x 10(6)/kg, n = 13), a statistically significant but moderate decrease in the duration of neutropenia and thrombocytopenia was observed in patients who received the highest doses of CD34+ cells. Our results confirm the utility of measuring the number of the CD34+ cells in peripheral blood as well as in the product collected by apheresis, but they challenge the place of cultures of progenitor haematopoietic cells. Moreover, the infusion of large amounts of CD34+ cells shortens the duration of the aplasia.

OSTEOLYSIS AND HYPERCALCEMIA: Multiple myeloma is a type B high-grade lymphoproliferative syndrome with bone tropism. Bone-related manifestations--osteolysis and hypercalcemia--are observed in 80 and 30% of cases respectively. Excessive bone resorption subsequent to destruction of the bone matrix by osteoclasts is associated with insufficient bone formation. This process plays a determining role in the development of osteolysis and hypercalcemia in multiple myeloma patients. MECHANISM OF BONE DESTRUCTION: The reality and intensity of bone destruction is clearly demonstrated by histomorphometric studies and more recently by biochemical methods using markers of bone resorption. The excessive bone resorption results from complex interactions between tumor plasma cells, bone cells, and stem cells and involves local factors and adhesion molecules. BISPHOSPHONATES: Bisphosphonates are powerful inhibitors of bone resorption. They constitute a substantial advance in the management of bone manifestation in patients with multiple myeloma. Bisphosphonates not only have a well-established curative effect in patients with tumor-induced hypercalcemia, but also inhibit disease progression in bone.

Over the past ten years, knowledge regarding the origin of the oligodendrocyte lineage during development has increased considerably. In this review, we present the major findings as a result of which it was determined that only restricted regions of the neural tube have an oligodendrogenetic potential. These findings are based on the study of molecular markers permitting the detection, among the multipotent neutricular cells of the neural tube, of those with a potential to differentiate into oligodendrocytes. At the present time, it appears that these oligodendrocyte precursors can be distinguished in the brain either by the expression of the plp/dm-20 transcript, or by that of the alpha-receptor of PDGF. These two markers allow two oligodendroglial subpopulations to be differentiated, suggesting a multiple origin of oligodendrocytes. An assessment has also been made of the current state of knowledge, still incomplete, regarding the intrinsic and extrinsic factors which cause a multipotent cell strain to follow an oligodendroglial differentiation. A better knowledge of the oligodendrogenesis during embryonic development should provide insight into the regeneration mechanisms, and later to the development of therapeutic strategies aimed at increasing the endogenous potential of remyelinization.

Limbal autograft transplantation is the procedure of choice in the management of ocular surface disorders secondary to stem cells deficiency. The aim of our study was to investigate the indications, results and limits of this infrequent surgery.

Microscopy, cell staining methods and tests for characterization of coagulation factor deficiencies are the landmarks of hematology at the beginning. Direct access to bone marrow, automatization, development of in vitro culture systems, monoclonal antibodies, biochemistry of proteins and nucleic acids were used to build today conception of the blood. Definitions of stem cells, growth factors, chromosomal translocations in leukemias and coagulation cascade represent the various aspects of the complex scientific object that the blood became.

A phase I clinical trial is being currently performed in our institution, aiming at evaluating the feasibility and toxicity related to the administration of Herpes Simplex-thymidine kinase gene-expressing human primary T lymphocytes following allogeneic hematopoietic stem cell transplantation. The need for safe and standardized preparation conditions for gene-modified cells is crucial. We describe the closed culture system used in the current trial for ex vivo retroviral-mediated gene transfer and transduced cell selection. Cell handling is performed in closed systems using sampling and transfer pack bags, culture bags and a sterile connection device which avoids opening the culture system. This closed system allows safe and reproducible ex vivo preparation of gene-modified primary T-lymphocytes for clinical use.

For the past thirty years, hematology has switched from the concept of bone marrow transplantation to the concept of hematopoietic stem cell (HSC) transplantation, from allograft to autograft, from non-manipulated graft to hyper-selection, from hematopoietic cellular therapy to immunotherapy. Indications of these transplantations are now more clear for malignant diseases and are ongoing for auto-immune diseases. A better knowledge of the HSC allows the control of their proliferation and differentiation, opening the field of ex vivo expansion. Very recently, new stem cells have been identified, establishing that a differentiated cell retain its totipotency: a nervous system cell can differentiate into HSC, which will further give hematopoiesis, mesenchymental cells or hepatocytes. New tools are under development: human ES cells, biomaterials, functionalized materials, opening the field of cellular engineering in the year 2000.

Neurological involvement in Behçet's disease, the cause of the disease's severe functional sequellae, is reported in 5.3 to 30% of cases. Coagulation disorders have been reported but they cannot explain the different thrombotic manifestations which are probably the consequence of an abnormal response of the vascular endothelial cells. Neurological manifestations include: a) dural sinus thrombosis which can be diagnosed by angio-MRI and whose prognosis is improved with the use of anticoagulants; b) exceptional lesions to arteries supplying the brain; c) aseptic meningitis and meningo-encephalitis; and d) exceptionally, solitary spinal cord involvement and peripheral disease. Neurological involvement can occur early or late after development of skin and mucosal signs and when inaugural make mislead diagnosis. The spinal tap usually gives objective evidence of lymphocyte meningitis. MRI is nonspecific, but the T2 and Flair sequences can evidence hypersignal areas, preferentially in the brain stem, basal nuclei, and subtentorial white matter with no preference for the periventricular regions. Spontaneous aggravation is the rule and the neurological prognosis is severe (dementia, pseudo-bulbar syndrome, loss of independence). Treatment is similar to that used for vasculitis and is aimed initially at reducing the inflammation with corticosteroids and at preventing relapse with the adjunction of an immunosuppressor. Results are better when treatment begins early; restitutio ad integrum has been observed. Duration of treatment is poorly defined: immunosuppressors have been proposed for a minimal duration of 2 years; corticosteroid therapy can be tapered off but interruption would expose to relapse. A maintenance therapy is advisable and, in our opinion, should be proposed indefinitely combining colchicine (1 to 2 mg/d), anti-aggregate doses of aspirin, and low-dose corticosteroids (1/10 mg/kg/d).

CD34 SORTING: CD34 sorting is a means of selecting hematopoietic stem cells among heterogeneous populations of cells. The technique is based on the fact that only hematopoietic stem cells (excepting a few tumoral cells) carry the CD34 antigen. Generally this type of manipulation is performed in case of stem cell grafts or for the treatment of certain malignant hematological diseases or certain solid tumors.

Engraftment in relation to infused CD34+ cell number was retrospectively analysed in 66 patients with hematological diseases: non-Hodgkin's lymphoma (n = 33), multiple myeloma (n = 21), acute myelogenous leukemia (n = 7), Hodgkin's disease (n = 4) and myelodysplastic syndrome (n = 1). Progenitor cells were mobilized with rhG-CSF, alone or in association with chemotherapy. The cells were harvested by leukapheresis until at least 2 x 10(6) CD34+/kg body weight were obtained. A total of 194 leukaphereses were performed (median = 3 per patient, range 1-9). A median of 3.40 x 10(8) nucleated cells/kg (range 0.31-27.59) and a median of 7.15 x 10(6) CD34+ cell/kg (range 1.31-115.70) were transplanted. Regardless of transfusional support or patient diagnosis, engraftment was rapid in patients who had received > or = 5 x 10(6) CD34+ cell/kg. In this case, absolute neutrophil blood count > or = 0.5 x 10(9)/l was obtained on day 12 post graft (range 7-19) and platelet count > or = 20 x 10(9)/l was also reached after the same median time interval (range 8-121). From the present results, a minimal threshold of 5 x 10(6) CD34+ cell/kg appears to be suitable for providing rapid and complete hematopoieitc reconstitution in patients exposed to high doses of chemotherapy with or without total body irradiation. Furthermore, administration of rhG-CSF during post-graft period significantly decreased the neutrophil time recovery (P = 0.002) but not that of platelets (P > 0.05).

THE FRENCH LAW ON BIO-ETHICS BEING REVISITED: The GRET (Group on ethical aspects of transplantation) makes a series of proposals in order to update some articles of the law, this being considered as a considerable progress. BONE MARROW GRAF: The new concept of graft of "Hematopoietic stern cells" including their different sources, better reflects the current practice, remaining considered basically as an organ. ORGAN TRANSPLANTATION, WITH LIVING DONOR: The extension to new categories of donors is proposed, with, however, the creation of a multidisciplinary committee of experts and representatives of the society in charge of the evaluation of the motivations, with objective criteria. ORGAN TRANSPLANTATION, DECEASED DONOR: A better information of the public, together with the help of Associations is proposed, and the creation of help units in order to provide support for family members, who come in emergency as witnesses and greatly need support and help.

To display theorical and methodological basis of the molecular biology. To point out its main medical applications.

Using a mouse model, we examine drug targeting towards bone marrow. One cytotoxic (doxorubicin) and one stimulating (rhG-CSF), bound to polyalkylcyanoacrylate nanoparticles, were studied. Histological studies, using a fluorescence microscope, showed rapid capture of nanoparticles by bone marrow macrophages and granulocytes as soon as 15 minutes after injection into the blood stream. Doxorubicin nanoparticles, administered at a dose of 11 mg/kg were more toxic than free doxorubicin on all blood and marrow cell lines. Moreover, the choice of the nature of the polymer had an influence on toxicity: doxorubicin polyisohexylcyanoacrylate nanoparticles were more toxic than polyisobutylcyanoacrylate particles. Quantification of doxorubicin in bone marrow has confirmed these results. The bone marrow concentrations observed demonstrated that there was a high level of targeting towards the bone marrow that would be very interesting to use for a stimulating drug. Nevertheless, rhG-CSF nanoparticles did not show better efficacy than free rhG-CSF.

The hypothesis that the endothelial and hemopoietic lineages have a common ontogenic origin is currently being revived. We have shown previously by means of quail/chick transplantations that two subsets of the mesoderm give rise to endothelial precursors: a dorsal one, the somite, produces pure angioblasts (angiopoietic potential), while a ventral one, the splanchnopleural mesoderm, gives rise to progenitors with a dual endothelial and hemopoietic potential (hemangiopoietic potential). To investigate the cellular and molecular controls of the angiopoietic/hemangiopoietic potential, we devised an in vivo assay based on the polarized homing of hemopoietic cell precursors to the floor of the aorta detectable in the quail/chick model. In the present work, quail mesoderm was grafted, after various pretreatments, onto the splanchnopleure of a chick host; the homing pattern and nature of graft-derived cells were analyzed thereafter using the QH1 monoclonal antibody which recognizes both quail endothelial and hemopoietic lineages. We report that transient contact with endoderm or ectoderm could change the behavior of cells derived from treated mesoderm, and that the effect of these germ layers could be mimicked by treatment with several growth factors VEGF, bFGF, TGF beta 1, EGF and TGF alpha, known to be involved in endothelial commitment and proliferation, and/or hemopoietic processes. The endoderm induced a hemangiopoietic potential in the associated mesoderm. Indeed, the association of paraxial or somatopleural mesoderm with endoderm promoted the "ventral homing" and the production of hemopoietic cells from mesoderm not normally endowed with this potential. The hemangiopoietic induction by endoderm could be mimicked by VEGF, bFGF and TGF beta 1. In contrast, a contact with ectoderm or EGF/TGF alpha treatments totally abrogated the hemangiopoietic capacity of the splanchnopleural mesoderm which produced pure angioblasts with no "ventral homing" behavior. We postulate that two gradients, one positive and one negative, modulate the angiopoietic/hemangiopoietic potential of the mesoderm.

We have investigated the developmental relationship of the hemopoietic and endothelial lineages in the floor of the chicken aorta, a site of hemopoietic progenitor emergence in the embryo proper. We show that, prior to the onset of hemopoiesis, the aortic endothelium uniformly expresses the endothelium-specific membrane receptor VEGF-R2. The onset of hemopoiesis can be precisely determined by detecting the common leukocyte antigen CD45. VEGF-R2 and CD45 are expressed in complementary fashion, namely the hemopoietic clusters in the floor of the aorta are CD45+/VEGF-R2-, while the rest of the aortic endothelium is CD45-/VEGF-R2+. To determine if the hemopoietic clusters are derived from EC, we tagged the E2 endothelial tree with a non-replicative retroviral vector and low density lipoproteins. Twenty four-48 hours later, labelled cells in the vascular tree were found to be either endothelial or hemopoietic but exceptionally both. Another 1-2 days later, groups of labelled cells appear in the dorsal mesentery within the hemopoietic "paraortic foci". Since no CD45+ cells were inserted among endothelial cells at the time of vascular labelling, hemopoietic clusters and foci must be concluded to derive from precursors with an endothelial phenotype.