The human hematopoiesis produces daily in the bone marrow several billions of blood cells. Bone marrow cells could be classified in three categories: the pluripotent stem cells able to self renewal and to differentiate in every kind of blood cell lineage, the progenitors, already engaged in one cell lineage and precursors, already recognizable on the bone marrow smear. This hematopoietic mechanism is controlled by a complex network of stimulating and inhibitory molecules. We report the interest of such basic research in the establishment of hematopoietic stem cell transplantation, their in vitro treatment and their selection. We point out the value of these laboratory technics for autografting monitoring.

Resistance is often defined as a lack of therapeutic response. Cellular resistance involves a decrease in intracellular levels of the antitumor agent due to a variety of mechanisms. These mechanisms are active in tumors with initial resistance as well as in those which respond initially but fail to be completely destroyed by chemotherapy. Although acquired forms of resistance seem to be the result of selection, some studies suggest that antitumor agents may induce resistance. Four main mechanisms of resistance are currently being investigated: 1) multidrug resistance, involving expression of a membrane P-glycoprotein, responsible for resistance to hydrophobic cationic agents; 2) detoxification of hydrophilic agents by the enzyme glutathione-S-transferase; 3) increased production of enzymes targeted by antimetabolites; 4) mutation or decreased synthesis of topoisomerases I and II which are the targets of very recent antitumor agents. New data were presented at the 1992 symposium of the American Association for Cancer Research; expression of P-glycoprotein is controlled by the mutant protein P53, the oncogene ras and differentiation agents. Physiological effects of this molecule are related to the chloride pump. Bone marrow stem cells from transgenic mice obtained by transfection of the gene MDR1 in germ cells exhibit resistance. Many agents can reverse P-glycoprotein-related resistance. Results from three phase I trials with Cyclosporin A as reversion agent were reported. It is essential to conduct clinical trials in order to assess the true value of these new data which hold promise for improving the performance of antitumor agents.

The aim of this review is to analyze the different ways by which stem cells and microenvironmental cells may interact. Stem cells are defined as immature cells that ensure the continuous renewal of blood cells. This small set of marrow cells comprises different kinds of cells, differing by their degree of maturity, their commitment, self-renewal ability and repopulating capacity. Microenvironmental cells are fixed marrow cells involved in stem cell survival, proliferation and differentiation. In vivo studies on the distribution within spleen or marrow, of stem cells injected to lethally irradiated mice, have suggested that cells of the microenvironment play a significant role in stem cell proliferation and differentiation. This role has been demonstrated using an in vitro model, i.e. the long-term marrow cultures as described in 1976 by M. Dexter. Analysis of stem cell maintenance in this culture system has made it possible to define the different means by which stromal cells and macrophages (the microenvironmental cells) may control stem cell behavior. Different molecules play a critical role: cytokines (growth factors and inhibitors), adhesion molecules (cell adhesion molecules and molecules belonging to the extracellular matrix) and eventually small peptides. It appears nowadays possible to materially represent the hemopoietic niche, whose existence was postulated by R. Schofield 10 years ago for theoretical reasons related to the the physiology of stem cells.

After a long gestation new therapies are at hand. At the present time about 30 patients have been cured with the bone marrow transplantation. The potential use of cord blood, rich in hematopoietic stem cells, is under development as an alternative for bone marrow transplantation. The activation of HbF expression upon hydroxyurea (Hydrea) and other agents which is under clinical trials. The agent 12C79 which increases the oxygen affinity of sickle cells in vivo and prevent HbS polymerization is in clinical development. Membrane acting agents which inhibit sickle cell dehydration are another approach to be developed. The inhibition of vascular cell adhesion, of inappropriate clotting formation and of inadequate response to vaso-occlusive events needs to be developed. Finally the gene therapy is a very promising avenue.

The development of immune defense mechanisms begins early during fetal life but is not yet completed at birth. The earliest hematopoietic stem cells, which give rise to the lymphocytic and myelomonocytic cell lines, appear between the fourth and eight weeks postconception. T cells, responsible for cellular immune defense mechanisms, are first detectable at 12 weeks and seem to acquire their functional capabilities at the 16th week. B lymphocytes, responsible for humoral immunity, can be identified in the fetal liver at eight weeks and are functional at the 12-th-13th week, with the ability to produce specific IgM antibodies. Thus, in theory, fetuses of more than 16 weeks can produce a full immune response. Neonates still have incomplete development of a number of mechanisms involved in nonspecific immunity (natural killer, polynuclear and macrophage chemo-attraction, complement system).

In 1992, the Melphalan-Prednisone (M. P.) protocol remains a standard treatment of multiple myeloma even if a lot of new ways have been investigated during the last years. Polychemotherapies may appear better than M.P. for high tumor mass myeloma. Interferon is useful as maintenance treatment after chemotherapy. Combinations of interferon and chemotherapy, during the induction phase, are under evaluation. Because of their toxicity, heavier treatments, with stem cells reinfusion, are being developed mainly with younger patients. Thanks to these approaches the response's rate is increasing but any improvement of survival is still to be demonstrated. Other recent investigations have concerned diphosphonates and immunoregulators. Larger use of these new treatments requires more informations about prognostic factors and their integration in therapeutic strategy of myeloma.

Peripheral blood and umbilical cord blood are promising sources of hemopoietic stem cells for autologous as well as allogeneic transplantation. The nature of the progenitors responsible for early marrow reconstitution after transplantation on one hand and for permanent reconstitution on the other hand is getting more precise. Features of the cells responsible for GVHD and of those responsible for GVL effect (graft versus leukemia effect) will soon allow a distinct monitoring of these two activities. Molecular biology will soon permit genetic labelling of the graft and thereby bring more precision for its follow-up and modulation in vivo in the post-graft period. Correction of an autologous transplant by introduction of the adequate gene in the hematopoietic stem cells in case of genetic anomaly at this level, should in the future, as suggested by animal studies, allow permanent correction of genetic disorders in patients autografted according to such a procedure. Finally, introduction of procedure like antisense nucleotides against DNA regions responsible for the malignant proliferation of tumoral cell, could extend the therapeutic possibilities of autograft.

Only in the last decade have autologous grafts begun to be studied extensively. Their most attractive feature is the avoidance of GVHD. However, GVHD has antitumoral effect on residual leukemic cells called "graft versus leukemia" effect and better understanding of this phenomenon explains the higher relapse rate after autologous bone marrow transplantation. New approaches such as cyclosporin--induced GVHD and IL-2 administration after autograft bring great expectations in this field. Colony stimulating factors and harvesting of peripheral stem cells help to reduce the duration of neutropenia. Finally, various techniques for marrow purging and hematopoietic cell isolation should make it possible to eliminate minimal residual disease. Recent results of autologous bone marrow transplantation in various malignancies are discussed.

In the light of recently published data, the mast cell can now be viewed as a key cell, not only in allergic reactions such as immediate hypersensitivity responses, but also in a broad spectrum of other biologic responses including host-parasite interactions, nonspecific inflammatory reactions, fibrosis, angiogenesis, tissue reconstruction, and wound healing. Nevertheless the molecular basis for the intervention of mast cells in many of these biologic responses is still unclear. Very recent studies have demonstrated that mast cells are capable of producing a wide range of cytokines, a property which may influence various physiologic, immunologic and disease processes. Furthermore, although substantial differences have been reported between mast cells located in different tissues, the reasons and mechanisms underlying this heterogeneity long remained obscure. The recent development of two original experimental approaches, i.e., in vitro culture of mast cells, mainly derived from mouse bone marrow precursors, and replenishment of mast cell-deficient mice, has provided new insight into the mechanisms by which tissue microenvironment influence of regulation mast cell phenotype. Extrapolation to humans of data obtained in rodents is, however, hazardous. In the review presented here, the most recent data from the literature provide the basis for outlining avenues of research which can be expected, in the near or remote future, to solve what mast cell experts term "the riddle of the mast cells".

A 52-year old man had a generalized seizure followed by progressive memory disturbances, affective changes, right hemiplegia and aphasia. He died 4 years later after a period of coma. Neuropathological findings included slight cortical atrophy, pallor of the centrum ovale, and infiltration of the cortex and subcortical white matter by neoplastic glial cells, with neither major neuronal loss nor spongiosis. Microglial rod cells were observed. The gliomatosis extended within the thalamus and subthalamic area on both sides, whereas the brain stem was much less involved. The spinal cord and peripheral nerves were not examined. Abnormal glial cells were stained by the glial fibrillary acid protein, which confirms the astrocytic differentiation of the tumoral cells.

3'-azido-3'-deoxythymidine (Azidothymidine or AZT) has attained wide critical utility in the treatment of acquired immunodeficiency syndrome (AIDS). Unfortunately, treatment with AZT is associated with the development of severe hematopoietic toxicity. The AZT sensitivity of marrow progenitors was different with an IC 50 of 10(-8) M and 10(-6) M for respectively BFU-E and CFU-GM/GEMM. Data reported here show that recombinant human interleukin-1 alpha (IL-1 alpha), a pleiotropic cytokine, was demonstrated to be efficient to protect normal human as well as murine hematopoietic progenitors (CFU-GM, CFU-GEMM and BFU-E) from the toxic effect of AZT. The maximal effect was observed with 30 U/ml (Human cells) or 100 U/ml (murine cells) IL-1 alpha for BFU-E and CFU-GM/GEMM, with a marked effect at 1 U/ml. The results demonstrate that marrow progenitors respond differently to AZT and point out the potential efficacy of IL-1 alpha to enhance the proliferation of hematopoietic stem cells treated with growth factors (IL-3, erythropoietin) and to minimize the hematopoietic toxicity associated with AZT treatment.

The application of gene transfer technology to human gene therapy has been intensively investigated during the last decade. The first human clinical trials in adenosine deaminase deficiency and metastatic melanoma recently demonstrated the feasibility and safety of using retroviral gene transduction for human gene therapy. Many problems remain to be solved for a better understanding of the functioning of genes to be transferred, a better efficiency of gene transfer and genetic correction by site-specific targeting in vivo, a better knowledge of the biological properties of target cells such as totipotent hematopoïetic stem cells. Clinical developments are expected in genetic diseases (immunodeficiency, thalassemia, hemophilia) and non genetic disorders (lymphokine gene therapy for cancer).

There are now rather straightforward methods to create transgenic animals whose genome is altered at the germline level. One method consists in the micro-injection of a gene into the pronucleus of a fertilized egg, the second one involves an homologous recombination event obtained in embryonic stem cells in culture. Only the latter method could eventually lead to an authentic gene therapy since it could actually substitute a normal gene for a mutated one instead of merely introducing a supplementary gene as done by micro-injection. Description of these techniques makes it obvious that germline therapy of human beings would not only be inacceptable on ethical grounds but would also hardly have any medical indications. Quite on the contrary, somatic gene therapy does not suffer from the same reservations and has numerous potential applications to man. As a matter of fact, several protocols have already received approval and have reached the stage of clinical trials: for example SCID (severe combined immunodeficiency due to a mutated adenosine deaminase gene), AIDS as well as some forms of malignant tumors.

Seraspenide, a synthetic tetrapeptide, inhibits cell cycle entry of normal hematopoietic stem cells. In mice it protects hemopoiesis against the damage caused by cytarabine, cyclophosphamide and carboplatin. Seraspenide has been given to 53 cancer patients undergoing monochemotherapy with cytarabine and ifosfamide in a double-blind cross-over randomized study. A significant protection of peripheral blood cells has been observed. Seraspenide has been devoided of toxicity.

The intra and extracellular kinetics of AcSDKP levels in cell culture have been studied. A close correlation was observed between the minimal level of intracellular AcSDKP (a negative regulator of cell proliferation) and the initiation of DNA synthesis. The return to initial levels of intracellular AcSDKP when the rate of DNA synthesis decreases, suggests a role for the tetrapeptide in homeostasis during cell growth. The return to normal values was not observed in preliminary studies on cell lineages during uncontrolled proliferation.

Antibodies directed against the central nervous system were looked for by indirect immunohistochemistry in the sera of 8 patients with paraneoplastic neurological syndrome (group 1), 21 cancer patients without neurological signs, 23 patients with miscellaneous neurological diseases and 63 normal subjects (groups 2 to 4). Four patients in group 1 had very high titres of antibodies. In 2 patients with small-cell lung carcinoma associated with sensory neuropathy the antibody recognized the cytoplasm and nucleus of all neurons. A 37 Kd protein was recognized by Western blot. A woman with cancer of the ovary and cerebellar syndrome exhibited an antibody against Purkinje's cell cytoplasm with a band of about 50-55 Kd at Western blot. In a woman with chronic uveitis and cerebellar atrophy with disappearance of Purkinje's cells the antibody (in blood and CSF) recognized certain layers of the retina as well as glial cells and cells present in the subependymal areas of the brain. Two bands of 46 and 59 Kd were revealed by Western blot. Immunoglobulins were detected in the cytoplasm of white matter cells in the cerebellum and brain stem. Among the other groups, one patient with lung cancer had a moderate titre of neuronal antinuclear antibody. The Western blot test was negative. The relevance of these antibodies for the diagnosis and treatment is discussed.