Ontogenic emergence of the hematon, a morphogenetic stromal unit that supports multipotential hematopoietic progenitors in mouse bone marrow.

Development of the full repertoire of hematopoietic-lymphopoietic cells from a single stem cell requires specific contacts with stromal cells. The spatio-temporal organization of these cell associations in the bone marrow in ontogeny is, however, not well understood. In the adult, 10% of marrow cells form a cohort of compact aggregates, the hematon. In the hematon mesenchymal cells (Stro-1(+)), perivascular lipocytes (desmin(+)), endothelial cells (CD34(+), Flk-1(+), Sca-1(+)), and macrophages amalgamate with the hematopoietic progenitors long-term culture-initiating cells (LTC-IC), cobblestone area-forming cell (CAFC), high-proliferative-potential colony-forming unit (HPP-CFU), granulocyte-macrophage (GM)-CFU, and burst-forming unit-erythroid (BFU-E). During endochondral ossification of the femur, GM-CFU and day 7 CAFC numbers increased progressively from day 17 of gestation, but primitive, day 35 LTC-IC appeared from postnatal day 2. Unexpectedly, bone marrow (BM) taken between embryonic day 17 and day 5 was unable to support myeloid cell production in long-term cultures or to support day 35 LTC-IC growth. However, a gain in stromal cell competence occurred between days 7 and 10, which was correlated with the emergence of hematon in the BM. Thus, acquisition of hematopoietic competence by BM lags behind for approximately 10 days after the initial hematopoietic cell influx. In the adult, the hematon fraction was 3.7-fold enriched in day 35 LTC-IC over the buffy coat. It produced more GM-CFU and HPP-CFU in myeloid culture and more B cells in lymphopoietic "switch" cultures. It is reported that stromal hematopoietic units named hematons are specific morphogenetic structures that emerge at a well-defined postnatal stage of development in long bones, delineate discrete territories for hematopoietic stem cell seeding and development, embody the most productive hematogenous compartment in the BM, and probably enclose a morphogenetic organizer. (Blood. 2000;96:3763-3771)

Large scale recovery and characterization of stromal cell-associated primitive haemopoietic progenitor cells from filter-retained human bone marrow.

Bone marrow aspirates are composed of two cellular compartments, an abundant buffy coat suspension and a minor particulate fraction. The particulate fraction is routinely removed by filtration prior to transplantation in order to reduce the risk of embolism. This study shows that the filter-retained fraction includes many multicellular complexes, previously defined as haematons. A haematon is a finely arborized stromal-web which is tightly packed with haemopoietic progenitor cells and differentiated postmitotic cells. Comparison of the pooled buffy coat and the filter-retained materials from healthy donors showed that the haematon fraction contained 8-40 x 10(6) CD34+ cells, 20-115 x 10(3) high proliferative potential colony-forming cells (HPP-CFC) and 0.49-2.67 x 10(6) granulocyte-macrophage colony-forming unit (GM-CFU) which constituted 24+/-8% (10-36; n=8) of the total GM-CFU population harvested. Similar, but more variable recoveries of GM-CFU were obtained from the haematon fractions from patients with breast cancer (21+/-13%; n=10), Hodgkin's disease (33+/-19%; n=4), non-Hodgkin's lymphoma (21+/-18; n=7), but the recovery was lower from patients with acute myelogenous leukaemia (AML) (13+/-13%; n=6). The haematon fraction was enriched in CD34+ cells (2.5-fold), long-term culture initiating cells (LTC-IC/CAFC, week 5) (3.5-fold), HPP-CFC (2.8-fold) and GM-CFU (2.3-fold) over the buffy coat. Purified CD34+ cells expanded exponentially and produced 800 to 4000-fold more nucleated cells, 300 to 3500-fold more GM-CFU and 10 to 80-fold more HPP-CFC in stroma-free suspension culture with interleukin-1 (IL-1beta), IL-3, IL-6, GM-CSF and stem cell factor (SCF), than did the starting cell input. The haematon fraction produced significantly more progenitor cells than the buffy coat in long-term liquid culture (LTC). This was due to the higher frequency of LTC-IC/CAFC and to the presence of the whole spectrum of native, stroma cell-associated CAFC in haematons. Thus, the haematon includes the most productive haematogenous compartment in human BM. This simple enrichment strategy, using filter-retained haematons, provides a rational source of BM cells for large scale experimental and/or clinical studies on haemopoietic stem cells and on critical accessory stromal cells.

Hepatocellular carcinoma cell lines from diethylnitrosamine phenobarbital-treated rats. Characterization and sensitivity to endothall, a protein serine/threonine phosphatase-2A inhibitor.

Primary hepatocellular carcinoma (HCC) is probably one of the most common fatal forms of liver cancer. We have established permanent cell lines from diethylnitrosamine/phenobarbital induced primary rat liver carcinomas to study new anticancer therapies. The rat hepatocellular carcinoma cell lines (HR-2, HR-3, and HR-4) have been maintained in culture for over 3 years. They form tumors when transplanted sc or im into young syngeneic rats. Immunocytology (alpha-fetoprotein, albumin), biochemical (gamma-glutamyl transferase), and histochemical (glycogen) marker studies and electron microscopy (biliary canaliculi) showed unique, stable differentiation patterns in these tumor lines. They overproduced the c-met protooncogene product and formed colonies spontaneously in semisolid culture with high cloning efficiency (HR-2: 50%-80%, HR-3: 35%-50% and HR-4: 50%-65%). The sensitivity of these cell lines to inhibitors of protein ser/thr phosphatase-2A (PP2A), a key enzyme in the control of G1/S and G2/M cell cycle phase transitions in eukaryotes, was studied in vitro. The specific, weak inhibitor of PP2A, endothall, caused dose- and time-dependent cytostasis specifically in G2/M. The cells died later by apoptosis, which was confirmed by cytology (annexin V-FITC labeling, propidium iodide painting of apoptotic bodies) and by fluorescent activated cell sorter (FACS) DNA measurements. The HR-2, HR-3, HR-4, and Zajdela hepatocellular carcinomas were most sensitive to endothall (IC50 of 1.7, 1.2, 0.9, and 1.7 microg/mL), whereas newborn rat hepatocytes growing exponentially in primary culture (IC50 = 6.2 microg/mL), rat DHD/K12 colon carcinoma cells (IC50 = 3.6 microg/mL), or human HT-29 colon carcinoma cells (IC50 = 4.9 microg/mL) were less sensitive. Thus, endothall inhibits preferentially HCC growth and these new rat hepatocellular carcinoma lines may be useful for further biochemical and pharmacological studies on PP2A inhibitors, and for testing new forms of treatment of hepatic cell carcinomas.

Bone marrow stromal cell defects and 1 alpha,25-dihydroxyvitamin D3 deficiency underlying human myeloid leukemias.

Primary myelodysplasia (MDP) and acute and chronic myelogenous leukemias (AML, CML) are considered disorders of clonal stem cell division. Several constitutive gene defects that contribute to the development of abnormal cell behavior have been identified in the hematopoietic cells. The role of bone marrow stroma cells in leukemogenesis, however, has not been established. We studied the organization of the bone marrow (BM) microenvironment to see if it was impaired during the initiation and progression of these malignancies. The buffy coat, hematon, and plasma fractions were separated from BM aspirates taken from healthy donors and diseased subjects at distinct clinical stages. The structural integrity of the BM microenvironment was evaluated analyzing the morphogenetic unit, the hematon. The hematon is a multicellular complex that includes fibroblasts, adipocytes, endothelial cells, resident macrophages, hematopoietic cobblestone area-forming cells (CAFC), high-proliferative potential colony-forming cells (HPP-CFC), granulocyte-macrophage colony-forming unit (GM-CFU), burst-forming unit erythroid (BFU-E), and terminally differentiated cells in normal BM. Hematon complexes were present in most BM aspirates from healthy donors (46H+/55). But they were absent from most of the patients with MDP (21H+/62) and AML (5H+/24) in the first perceptible phase, and from those with CML throughout the disease (5H+/55). Hematon complexes were present in the BM aspirate in 22/36 AML patients at clinical remission after chemotherapy or differentiation therapy. The hematon fraction isolated from normal BM, contained 25 times more 25-hydroxyvitamin D3 and about 500-fold more 1alpha,25-dihydroxyvitamin D3 than the BM plasma. The concentration of 1alpha,25-dihydroxyvitamin D3 was low or undetectable in the BM plasma of some, but not all, patients with MDP (18/35) or AML (9/24). Thus, in the BM microenvironment, the metabolism of low-density lipids and lipophylic hormones are severely impaired prior to initiation or during the accelerated expansion of leukemia cells. The lack of organized stromal network and the decreased level of some lipophylic hormones, acting probably as morphogens, may contribute to the onset and progression of human myeloid leukemias.

Effects of norcantharidin, a protein phosphatase type-2A inhibitor, on the growth of normal and malignant haemopoietic cells.

Cantharidin is a natural toxin that inhibits protein phosphatase type 2A (PP2A) and has antitumour effects in man. We have studied the synthetic analogue, norcantharidin (NCTD), which has less nephrotoxic and phlogogenic side-effects, investigating the effects on the normal haemopoietic system and leukaemia cell growth. Daily intraperitoneal (i.p.) injection of NCTD induced dose and circadian time-dependent transient leucocytosis in normal mice, but did not accelerate bone marrow (BM) regeneration, or have haemopoietic offe-effects following chronic administration. NCTD stimulated the cell cycle progression of granulocyte-macrophage colony-forming cells (GM-CFC), stimulated DNA synthesis and increased the frequency of mitotic cells in short-term human BM cultures. NCTD also stimulated the production of interleukin (IL)-1 beta, colony stimulating activity (CSA) and tumour necrosis factor (TNF)-alpha. Continuous in vitro NCTD treatment, however, inhibited both DNA synthesis and GM-CFC growth. Fluorescence-activated cell sorting (FACS) analysis of DNA profiles and cytological studies in HL-60, K-562 or MRC5V2 (fibroblast) cells indicated that low doses of NCTD accelerated the G1/S phase transition, while higher doses or prolonged incubations inhibited the cell cycle at the G2/M phases or during the formation of postmitotic daughter cells. Electron microscopy revealed that NCTD impaired the neogenesis of chromatin material and nuclear membrane during the M/G1 phase transition in K-562 cells. The biphasic effect of NCTD may be due to inhibition of PP2A activity, which regulates the cell cycle, both at the restriction point and at the G2 and M phases. Our data provide new insight into the cellular and molecular actions of NCTD, and partly explain its therapeutical effects in cancer patients.

The hematon, a morphogenetic functional complex in mammalian bone marrow, involves erythroblastic islands and granulocytic cobblestones.

The development of pluripotential hematopoietic stem cells (PHSC) requires the continuous support provided by the bone tissue and bone marrow (BM) stromal cells. The basic rule of spatial and temporal organization of the distinct stromal cells and differentiating hematopoietic cells in the course of development, regenerative morphogenesis, or under homeostasis is still poorly understood. We have identified a cohort of preformed, multicellular aggregates in human BM aspirates that we have called hematons. This study shows that homologous hematon complexes can be isolated from the femoral BM shaft of normal mice. Cytologic analysis showed that both human and mouse hematons contained finely arborized endothelial cells, fibroblasts, preadipocytes, lipid-laden cells, and resident macrophages. This stromal cell web was tightly packed with hematopoietic cells comprising primitive cells with marrow-repopulating ability (MRA); day-8 and -12 colony-forming unit-spleen (CFU-S8 and -S12) in the mouse hematon; and high proliferative potential colony-forming cell (HPP-CFC), burst-forming unit-erythroid (BFU-E), granulocyte-macrophage-CFU (GM-CFU), as well as differentiated postmitotic cell populations in both human and mouse hematons. A cohort of single hematons produced a large, but variable, number of myeloid and erythroid cells, as well as megakaryocytes, in organotypic microculture, indicating the heterogenous growth potential of individual hematons. Each hematon developed into a complex, adherent colony in long-term liquid culture, which involves erythroblastic islands and granulocytic cobblestones. The hematons, isolated from 5-fluorouracil (5-FU)-treated mice, contained more HPP-CFC, BFU-E, and GM-CFU populations than the buffy coat (BC) fraction and produced significantly more CFU than normal hematons in organotypic microcultures. The present results provide further support for our hypothesis that the hematon is a tissue-specific complex structure that plays a critical roles in the maintenance of homeostasis and in the regenerative morphogenesis of mammalian BM.

Innate chaos: I. The origin and genesis of complex morphologies and homeotic regulation.

The genesis of complex morphologies is an inherent property of all dynamically expanding natural systems. In the inorganic and prebiotic world, chaotic movement of quantitable particles results in formation of ordered streamlined structures or micelles close to phase boundaries. In the course of chemical and colloid crystallization or development of living organisms, complex morphologies emerge, due to unusual chaotic attraction, diffusion limited aggregation (DLA) and multifractal organization suggesting that common mechanisms direct the morphogenesis in a wide range of natural systems. The development of a multicellular organism from a single fertilized oocyte requires intensive clonal proliferation sequential determinations and the organization of terminally differentiated cells in morphologically stable homeostatic functional units. Comparative data on insect and vertebrate embryogenesis revealed that the spatial organization of the developing body is orchestrated by several mechanisms: maternal effect genes or cell position specify the initial polarities and the main axes, while metameric segmentation, intrasegment identity and cell fate are determined by the programmed expression of morphogenetic determinants. They include evolutionarily conserved DNA binding proteins containing homeobox or pair-box sequences, endogenous ligands, activating specific nuclear hormone receptors, and humoral growth factors acting via specific membrane receptors and more ubiquitous transducing pathways. Morphogenetic regulators form intratissual gradients and demark fields required for the correct realization of the developmental programme. It has been recognized that the cell's freedom is limited to stringent developmental choices that in the end results in the formation of coherent cell colonies, many of them displaying chaotic behaviour. The linkage between embryonic regulation and adult tissue differentiation is not completely elucidated, however, data are emerging to show that several morphogenetic regulators may function throughout life in different human tissues. Genetically transmissible deletions or acquired impairments likely contribute to malignant tissue growth. Diffusible morphogenetic regulators may reverse the malignant phenotype in some cases and induce clinical remission. Further work is needed, however, to identify the dominant components of physiological regulatory networks and to understand what hierarchical organization and chaotic behaviour represent in order to elaborate new combined therapeutic protocols.

Combined differentiation therapy in myelodysplastic syndrome with retinoid acid, 1 alpha,25 dihydroxyvitamin D3, and prednisone.

The myelodysplastic syndrome (MDPS) provides an opportunity for identifying host factors (genetic, endocrine, immune) involved in initiation and progression of preleukemia into frank acute myeloid leukemia. The aim of this study was to identify bone marrow (BM) cellular and humoral dysfunctions central to the development of MDPS and useful in therapeutic follow-up studies. Our preclinical studies have shown that (1) the characteristic stromal cell composition of the normal BM microenvironment was impaired in MDPS and in AML in 67 and 86% of the cases, respectively; (2) the 1 alpha,25(OH)2D3 concentration in BM plasma was abnormal in 50% of MDPS and 30% of AML; and (3) an inverse correlation existed in MDPS between the 1 alpha,25(OH)2D3 concentration and the frequency of F-CFU, (r = 0.41, p < 0.02), suggestive of a regulatory interaction between this secosteroid hormone and BM stromal cells. The analysis of clonal extinction of BM blast cells in response to all trans retinoic acid (RA), 1 alpha,25(OH)2D3, and colony stimulating factors (PHA-LCM), either alone or in various combinations, revealed individual patterns of responses in the cases of MDPS or AML. The results indicate the necessity for preclinical studies to select patients for combined differentiation therapy. Our ongoing clinical trials suggest that RA (Roaccutan, 20 mg/day continuously) as induction therapy, followed at weeks 6 to 8 by prednisone (40 mg/day for 15 days) and 1 alpha,25(OH)2D3 (Rocaltrol, 3 x 0.25 micrograms/day for 3 months) may induce a long-lasting hematological remission in MDPS.

Roles for the heliodynamic hormones, all trans retinoic acid and 1 alpha, 25-dihydroxyvitamin D3, in control of the hematopoietic cell cycle.

It is now well established that the production of primary hematopoietic cells is controlled at different levels of the biological organization. Bone marrow (BM) stromal cells, the extracellular matrix (ECM), polypeptide hematopoietic growth factors (HGF) as well as endogenous cell-division cycle (CDC) related factors play a dominant role in this control. Recent information suggest that the 2 lipophilic hormones, transRA and 1 alpha,25D3, depending on and/or perhaps mediating solar energy, play a role in the maintenance of BM homeostasis. Here we show that both transRA and 1 alpha,25D3: a) modulate the growth and/or stimulate the adipocytic differentiation of fibroblastic stromal cells (F-CFU); b) inhibit the synthesis and extracellular processing but stimulate the solubilization of matrix collagen; c) modulate the clonal growth of myeloid progenitor cells (GM-CFU) in synergy with HGFs; and d) inhibit the production of lactic acid in standard, normal long-term BM cultures (LTBMC). Comparative analysis of normal, preleukemic and leukemic BM cells in LTBMC indicated a positive correlation between the induction of terminal differentiation and reduced lactate production elicited by transRA or 1 alpha,25D3. These results raise a hypothesis according to which the terminal differentiation induced by the helicodynamic hormones is dependent on the mitochondrial aerobic ATP-generating system whose impairment may be a critical step during the process of leukemic transformation.

Retinoic acid in mono- or combined differentiation therapy of myelodysplasia and acute promyelocytic leukemia.

Myelodysplastic preleukemic syndromes (MDPS) and acute promyelocytic leukemia (APL) share a surprising in vivo sensitivity to the hormonally acting 13 cis or all trans retinoic acids (transRA). Here we show that transRA as a monotherapeutic agent induced a stable remission in APL at the third relapse. In MDPS, treatment with prednisone and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25D3) 13 cis RA induced a long-lasting hematological remission. Initially both patients had an impaired BM microenvironment which regenerated on retinoid therapy as judged by reappearance of the Hematon fraction in the BM aspirates. Our preclinical experiments using long-term liquid BM cultures (LTBMC) indicated that several individual patterns of growth and differentiation responses can be induced by combinations of transRA, 1 alpha,25D3 and hemopoietic growth factors (HGFs). The biological responses may vary from complete clonal extinction to a significant growth stimulation of the leukemic blast cell populations. These results further support the importance of preclinical studies in selecting "good" responders for, and excluding "poor" responders from protocols using differentiation therapy.

Modulation of bone marrow cell functions in vitro by bestatin (ubenimex).

Bestatin (ubenimex), the microbial leucil-aminopeptidase B inhibitor, has been shown previously to stimulate both interleukin-1 (IL-1) and IL-2 production and to enhance T-cell, as well as macrophage mediated immunoreaction when administered in vivo in mice. Here we show that although Bestatin has no direct growth stimulatory activity, it enhances the growth of GM-CFU populations in semisolide culture and stimulates the cell production in liquide organotypic Hematon cultures in synergy with recombinant human GM-CSF. In long term human bone marrow culture Bestatin accelerated the adipocytic differentiation among colony forming stroma cells (F-CFU). Our data provide further evidences that Bestatin may interact with the hemopoietic cell renewal system at different levels of biological organisation.

Elliptinium, norcantharidin and tetrachlordecaoxide in combined chemo-immunotherapy prolong the survival of Friend-virus-infected mice.

The association of several drugs with different target specificities has long been proven to be more efficient than one single agent in the treatment of cancer. This strategy might also be of benefit in the cure of retrovirus-induced diseases. The effectiveness of several drug combinations was evaluated on DBA/2 mice injected with Friend leukaemia virus (FLV). Elliptinium (ELP), a known chemotherapeutic agent with possible antiviral activity, was given in association with norcantharidin (NCTD) (shown previously to increase the cytotoxic potential of human lymphocytes) and/or in association with tetrachlordecaoxide (TCDO), which augments both humoral and cellular immune responses. ELP alone at a dose of 0.2 mg/kg in long-term treatment significantly increased the survival of mice infected by FLV. When ELP, TCDO (2 micrograms/kg) and NCTD (2 mg/kg) were given together, the survival time was prolonged 1.6 times and 2 times as compared to the group treated by ELP alone or to non-treated controls, respectively. Moreover, the combined treatment gave more effective inhibition of hepatomegaly than ELP alone, suggesting that this protocol might have an organ-specific effect and might suppress the leukaemogenesis induced by FLV in some erythropoietic organs. These results indicate that a chemotherapeutic agent (ELP) associated with two immunomodulatory substances (NCTD and TCDO) is more effective than chemotherapy alone in controlling retroviral infection and in the prolongation of survival. These data taken together suggest that the role of the two immunomodulatory agents might be to suppress the retroviral infection synergistically with ELP and enhance immune functions. Possible modes of action are discussed and are under investigation.

Hematon, a multicellular functional unit in normal human bone marrow: structural organization, hemopoietic activity, and its relationship to myelodysplasia and myeloid leukemias.

An increasing amount of data provides strong evidence for the complex multifactorial control of primary hemopoietic functions. Here we present a new multicellular functional unit, the Hematon, isolated from the light-density floating fraction of normal human bone marrow (BM) aspirates. The Hematon is organized in a compact, three-dimensional spheroid complex from central adipocytes, fibroblastoid cells, and resident macrophages that compartmentalize myeloid, erythroid, and megakaryocyte progenitor cells and their progenies. The Hematon fraction is more than twofold more abundant in progenitor cells when compared to the mononuclear cell (MNC) fraction as gauged by cytological techniques and by analysis of granulocyte-macrophage colony-forming unit (GM-CFU) populations. Individual Hematons may produce, within 2-3 weeks, up to 50,000 hemopoietic cells of different cell lineages in organotypic microcultures. Recombinant human hematopoietic growth factors interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and macrophage colony-stimulating factor (M-CSF) significantly stimulated the endogenous cell production of some but not all of the individually treated Hematons, indicating the heterogeneity of factor-responsive cells within the Hematon population. Comparative observations of 184 BM aspirates support the hypothesis that the presence of Hematons in a BM aspirate correlates positively with homeostatic blood cell production, because the Hematon was present in normal BM (31/40) and it was rare among patients with myelodysplastic syndromes (15/53), acute myeloblastic leukemia (7/39), and chronic myelocytic leukemia (5/52). We suggest that the Hematon represents a unifying model around which the variability of fundamental BM functions and dysfunctions can be explored.

An ultrastructural study of normal human thymic epithelium in primary cultures.

The ultrastructural study of a large series of samples of normal human thymic epithelial cells (TECs) in primary cultures is here presented. Cultured TECs are heterogeneous due to marked differences in their cytoplasmic organization. In particular, a spectrum of ultrastructural features have been observed, ranging from poorly-differentiated cells to secretory and epidermoid-like ones, as well as intermediate transitional forms and cells co-expressing both epidermoid-like and secretory features. Cells with peculiar secretory (neuroendocrine-like) characteristics have also been identified in the culture obtained from one of the donors. These data suggest the existence of a thymic epithelial cell type which may act as a dynamic unit, changing its cytoplasmic organization in response to microenvironmental signals. The types and interrelationships of cytoplasmic organelles observed in the secretory cells suggest that the production of thymic peptides involves the classical pathway of protein synthesis and secretion.

Pathobiology of myelodysplastic syndromes.

The myelodysplastic/preleukemic syndromes represent unique clinical situations since patients with initially mild hemopoietic abnormalities can be singled out from those progressing into frank myeloid leukemia. Here we confront data focused on the identification of critical cellular, molecular biological, cytogenetic and physiological defects leading to leukemic progression. An increasing amount of data supports our earlier hypothesis according to which the impairment of an endogenous (intracellular) life-cycle suppressor gene-product, or functionally related regulatory genes, plays the decisive role in the course of disease progression. The identification of systemic as well as clonally transmissible defects have clinical importance since in some cases the therapeutic application of the appropriate physiological substances may result in long lasting hematological remission.

The differentiation-promoting potential of a cytostatic fluoro-pyranosyl adriamycin analog (FAD 104).

Acute toxicity to the hematopoietic cell renewal system is a critical side effect of most anticancer agents. Here we compared the effects of FAD-104 to those of the parent compound adriamycin (ADM) and of epi-adriamycin (epi-ADM) on the growth and differentiation of normal as well as leukemic human myeloid progenitor cells. FAD-104 was less toxic to myeloid colony-forming cells (GM-CFU) than ADM or epi-ADM. In addition, FAD-104 but not ADM induced a clonal down-grading in both normal and leukemic blast cells, and it stimulated the terminal differentiation of myeloid leukemia cells. Therefore, FAD-104 may be useful in the treatment of some forms of myeloid leukemia.

Circadian and seasonal rhythms in murine bone marrow colony-forming cells affect tolerance for the anticancer agent 4'-O-tetrahydropyranyladriamycin (THP).

The proliferative status of both bone marrow myeloid precursor cells (GM-CFUc) and stromal cells from the microenvironment are organized along circadian and circannual time scales in male B6D2F1 mice. Such temporal structure profoundly affects the in vitro susceptibility of myeloid precursors for the anticancer agent, 4'-O-tetrahydropyranyl adriamycin. These results account in part for the large-amplitude circadian rhythms that characterize host tolerance for chemotherapy. They also provide evidence that the time of bone marrow sampling from a donor may influence its engraftment into the recipient.

Hematon: a multicellular functional unit in primary hematopoiesis.

Bone marrow aspirates from healthy donors contain a fraction of low density multicellular spheroids, 100-500 microns in diameter. They are organized in a three-dimensional network consisting of central preadipocytes/adipocytes, mesenchymal and reticular cells, and resident macrophages that are closely associated with myeloid, erythroid and megakaryocyte progenitor cells and with their progenies. These spheroids are 2- to 5- fold more abundant in progenitor cells compared with the whole bone marrow as estimated by monoclonal antibody markers My 10 and T 9, by analysis of granulocyte--macrophage colony forming cells (GM-CFC) and by cytological techniques. They produce terminally differentiated cells in organotypic microcultures. We suggest that a multicellular spheroid may represent the fundamental unit of primary hematopoiesis; we therefore name it hematon. Here we show that the presence of hematons in bone marrow aspirates correlated positively with homeostatic blood cell production: they were present in normal bone marrow (BM) (19/25), and absent in myelodysplasic syndromes (MDPS) (8/21), in acute nonlymphocytic leukemias (ANLL) (3/22) and in chronic myeloid leukemia (CML) (2/28). The hematons were recovered under hematological remission in MDPS and in ANLL, suggesting that they may be dispersed reversibly in certain disease conditions. The hematons represent a unifying model around which the variability in some bone marrow cell functions can be explored.

Excess of lympho-reticular cell complexes in the bone marrow linked to T cell mediated dysmyelopoiesis.

The term dysmyelopoietic syndrome (DMPS) covers a variety of closely related disorders with various etiological factors, and characterized by chronic (pan) cytopenias whose prognosis and treatment are still controversial. Despite the recent efforts to identify pathogens, effector cells and soluble cell products involved in the development of this syndrome only a few comprehensive experimental data are available, useful for elaboration of therapeutic regimens. We describe here a patient with DMPS who was refractory to inductive chemotherapy. Initial agar gel culture studies revealed the activity of hematopoiesis inhibitory T cells within the bone marrow that could be suppressed by prednisone both in vitro and in vivo. However another pathological cell features, the excess of an unusual lympho-reticular cell complexes was identified in long-term liquid cultures. These symbiotic cell complexes persisted throughout the disease, despite the prednisone induced hematological remission, suggesting their causative role in the disease, that more recently progressed towards acute myeloid leukemia.

From experimental to clinical attempts in immunorestoration with bestatin and zinc.

Severe impairment of the lymphopoietic cell renewal system is an important etiological factor of cancer development and it may be the consequence of massive radio and/or chemotherapeutic regimens. In a comparative study, we analysed the potential, systemic immunorestoratory capacity of bestatin, a microbial leucil-aminopeptidase inhibitor and of the ubiquitous trace element zinc. In vivo administration of bestatin in mice stimulated both Interleukin 1 and Interleukin 2 production, and enhanced T cell, B cell as well as macrophage mediated immunoreactions. In a phase II clinical trial on 41 patients with non-Hodgkin lymphoma, Hodgkin disease and solid tumors, bestatin treatment corrected the pathological frequency of both OKT4 and OKT8 lymphocyte subpopulations. Zinc-saturated transferrin had a significative stimulatory effect on the ongoing DNA synthesis of antigen activated human lymphocytes in culture. Oral administration of zinc-gluconate to patients who manifested a severe T cell subpopulation defect corrected preferentially the OKT8 suppressor/cytotoxic T cell unbalances. The clinical results obtained by both bestatin and zinc were observed only on a short-term, so further studies are needed to elaborate long lasting regiments and to establish whether these treatments have determinant influence on the underlying disease.