Granulopoiesis in long-term culture by marrow from mice with busulfan-induced chronic latent aplasia

Mice given high-dose busulfan therapy develop a chronic latent marrow aplasia characterized by normal peripheral blood neutrophil numbers, hematocrits and marrow cellularity but reduced numbers of pluripotent hemopoietic stem cells (CFU-s) and granulocyte-monocyte progenitor cells (CFU-gm). To study the pathogenesis of this lesion, bone marrow was propagated in long-term marrow cultures (LTMC). Small amounts of normal marrow readily established and sustained long-term granulopoiesis in vitro. In contrast, inocula of marrow from busulfan-treated animals containing three to five times as many stem and progenitor cells failed to establish long-term granulopoiesis in vitro. These results suggest that high-dose busulfan therapy produces a qualitative defect in either the hemopoietic stem cells, the stromal-forming elements, or both, rendering them incapable of establishing long-term granulopoiesis in vitro. Furthermore, mixing experiments employing normal and busulfan-damaged marrow demonstrate that this qualitative defect is not due to the emergence of a suppressor cell population. LTMC can show types of marrow damage not detectable by other techniques currently available and represent a powerful tool for studying latent bone marrow failure.     

Protection of 3'-azido-3'-deoxythymidine induced toxicity to murine hematopoietic progenitors (CFU-GM, BFU-E and CFU-MEG) with interleukin-1

3'-Azido-3'-deoxythymidine (AZT) has attained wide clinical utility in the treatment of acquired immunodeficiency syndrome (AIDS). Unfortunately, associated with AZT use, is the development of severe hematopoietic toxicity as manifested by anemia, neutropenia and overall bone marrow suppression. Interleukin-1 (IL-1), a cytokine, primarily produced by activated macrophages, has been involved in the control of hematopoiesis by acting synergistically with other hematopoietic growth factors, and has been demonstrated to be an effective agent in reducing the myelosuppression associated with the therapy for malignant disease. We report here the ability of recombinant human IL-1 alpha to protect normal murine hematopoietic progenitors (CFU-GM, BFU-E, and CFU-Meg) from the toxic effects of AZT. Following the determination of the LD50 dose for each progenitor, IL-1 was added in co-culture studies (10-1000 units; 0.001-1.0 micrograms/ml protein) with adherent cell depleted marrow. Marrow progenitors expressed differences in AZT sensitivity, e.g., BFU-E, LD50 5 x 10(-9)M; CFU-Meg, LD50 10(-7) M; CFU-GM, 5 x 10(-5) M respectively. IL-1 inhibited AZT induced toxicity. The maximum IL-1 dose effect was observed for CFU-GM and CFU-Meg at 300 units, 0.3 micrograms protein; however BFU-E required a dose of 600 units, 0.6 micrograms/ml protein to reverse the effects of AZT. These results demonstrate marrow progenitors respond differently to AZT and identifies the potential efficacy of IL-1 to minimize the hematopoietic toxicity associated with AZT treatment.     

Lymphohemopoiesis in culture is prevented by interaction with adherent bone marrow cells from mutant viable motheaten mice

Mice with the recessive "motheaten" (me) or "viable motheaten" (mev) mutations have severe immunologic disturbances and die at an early age. The function of hemopoietic progenitor cells and microenvironmental elements that regulate their growth and differentiation were studied in mev mice with two types of long term bone marrow cultures. Cells from bone marrow of homozygous defective mev/mev mice were non-productive under conditions that normally support replication of stem cells and production of neutrophil granulocytes. Similarly, in a different culture system, lymphocytes were produced from normal littermate, but not mev/mev bone marrow. Initial overgrowth of cells having macrophage-like characteristics occurred in both culture systems with marrow from defective mice. Co-cultures of normal and defective bone marrow cells were always non-productive. In contrast, supernatants of mev/mev bone marrow cultures did not have a detrimental effect on cultures of normal cells, implying that the suppression was cell-associated. Furthermore, there was no evidence for abnormal release of granulocyte or macrophage growth factors in mev bone marrow cultures. A small population of cells in mev/mev bone marrow cultures were morphologically similar to "stromal" cells that support lymphohemopoiesis. Certain culture strategies could be used to enrich for these. mev/mev stromal cells had affinity for normal lymphocytes; however, they did not support lymphocyte growth. The long term bone marrow cultures thus reveal an apparent imbalance in the regulatory mechanisms affected by these single gene mutations. This is manifested by preferential or aberrant growth of one type of adherent cell and a possible functional abnormality of stromal cells. mev mice could provide an ideal model for investigating cell-associated molecules that normally limit progenitor cell replication.     

Aplastic anemia,cellular and molecular aspects

Aplastic anemia (AA) is an autoimmune disorder characterized by bone marrow and peripheral blood pancytopenia. Different environmental and genetical conditions could be effective in an outbreak of this disease. The exact pathogenesis of this disease, however, is still idiopathic. The present study is based on Pubmed database information (2002–2021) using the words “Aplastic Anemia,” “Hematopoietic Stem Cells niche,” “Signaling pathway,” “Cytokines,” and “Immuno cells.” In this disease, both hematopoietic stem cells and mesenchymal stromal cells are impaired, which is associated with impaired hematopoiesis and decreased hematopoietic cells. Inflammatory cytokines increase, which changes the ratio of T lymphocytes and leads to disease progression. In addition, the most common mechanism of AA is damage by the immune system, which leads to increased apoptosis in progenitor cells. We have shown in this review that the disease involves quantitative defects in stem cell numbers and qualitative abnormalities in the function of these cells and the activity of many different cellular and molecular factors can damage hematopoietic cells and the protective substrate of these cells in this disease.     

Effects of human leukocyte conditioned medium on mouse haemopoietic progenitor cells.

Medium conditioned by human peripheral blood leukocytes (HLCM) was studied for its in vitro effects on haemopoietic progenitor cells (CFU-s and CFU-c) present in mouse bone marrow. HLCM has poor colony stimulating activity in semi-solid cultures of mouse bone marrow cells, but invariably increases the number of colonies obtained in the presence of plateau levels of semi-purified colony stimulating factor (CSF). In liquid cultures, HLCM appears to contain a potent initiator of DNA synthesis in CFU-s, an activity which coincides with an increased CFU-s maintenance and causes a three- to four-fold increase in CFU-c number. It is apparent from this study that HLCM, in addition to stimulating colony formation in cultures of human bone marrow cells, has a profound in vitro effect on primitive haemopoietic progenitor cells of the mouse, which cannot be attributed to CSF.     

Interleukin-3 induces proliferation but not lymphokine activated killer activity from human and murine mononuclear cells

Recombinant IL-3 (rIL-3) is a potent colony stimulating factor capable of stimulating early hematopoietic pluripotential progenitor cells and of supporting the differentiation of multiple cells. IL-3 has also been shown to have effects on mature, differentiated circulating cells including eosinophils and T cells. We evaluated the role of exogenous rIL-3 in the generation of cells with LAK activity from murine splenocytes and human bone marrow, spleen, unseparated PBMC and purified null cell preparations. rIL-3 was unable to generate lytic activity from any of these populations by itself and appeared to decrease LAK activity in bone marrow cultures containing high dose IL-2, (bone marrow derived cells (n = 3) with LAK activity for fresh tumor, mean lytic units(LU) 94.6 +/- 63.5 vs 32.8 +/- 44.8 for IL-2 and IL-2 plus IL-3 cultures, respectively p2 less than 0.05). Unlike previous reports testing murine cells, IL-3 priming and subsequent culture in IL-2 of human unseparated bone marrow cells or human or murine splenocytes, failed to generate long-term cultures with lytic activity. IL-3 did, however, induce a dose dependent stimulation of bone marrow and null cell preparations (mean null cell stimulation (3H Thymidine incorporation) with IL-3, 436 +/- 168 cpm vs 9802 +/- 9799 cpm, for 0 vs 10(3) units of IL-3, respectively n = 4, p2 less than 0.05). Furthermore, in bone marrow, unseparated PBMC and null cell cultures, the addition of rIL-3 generated characteristic large blastic appearing cells with prominent basophilic granules.(ABSTRACT TRUNCATED AT 250 WORDS)     

EFFECTS OF HUMAN LEUKOCYTE CONDITIONED MEDIUM ON MOUSE HAEMOPOIETIC PROGENITOR CELLS

Medium conditioned by human peripheral blood leukocytes (HLCM) was studied for its in vitro effects on haemopoietic progenitor cells (CFU-s and CFU-c) present in mouse bone marrow. HLCM has poor colony stimulating activity in semi-solid cultures of mouse bone marrow cells. but invariably increases the number of colonies obtained in the presence of plateau levels of semi-purified colony stimulating factor (CSF). In liquid cultures, HLCM appears to contain a potent initiator of DNA synthesis in CFU-s. an activity which coincides with an increased CFU-s maintenance and causes a three- to four-fold increase in CFU-c number. It is apparent from this study that HLCM, in addition to stimulating colony formation in cultures of human bone marrow cells, has a profound in vitro effect on primitive haemopoietic progenitor cells of the mouse, which cannot be attributed to CSF.     

Isolation and enrichment of skeletal muscle progenitor cells from mouse bone marrow

There is great interest in the therapeutic potential of non-hematopoietic stem cells obtained from bone marrow called mesenchymal stem cells (MSCs). Rare myogenic progenitor cells in MSC cultures have been shown to convert into skeletal muscle cells in vitro and also in vivo after transplantation of bone marrow into mice. To be clinically useful, however, isolation and expansion of myogenic progenitor cells is important to improve the efficacy of cell transplantation in generating normal skeletal muscle cells. We introduced into MSCs obtained from mouse bone marrow, a plasmid vector in which an antibiotic (Zeocin) resistance gene is driven by MyoD and Myf5 enhancer elements, which are selectively active in skeletal muscle progenitor cells. Myogenic precursor cells were then isolated by antibiotic selection, expanded in culture, and shown to differentiate appropriately into multinucleate myotubes in vitro. Our results show that using a genetic selection strategy, an enriched population of myogenic progenitor cells, which will be useful for cell transplantation therapies, can be isolated from MSCs.     

Influence of IL-1 alpha and -1 beta on the survival of human bone marrow cells responding to hematopoietic colony-stimulating factors

Purified recombinant human (rhu) IL-1 alpha and IL-1 beta were evaluated for their effects on the proliferation and survival of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells from normal human bone marrow (BM). Using nonadherent low density T lymphocyte depleted (NALT-) BM cells cultured in the presence or absence of IL-1, CSF-deprivation studies demonstrated that IL-1 alpha or IL-1 beta by itself did not enhance the proliferation of CFU-GM or BFU-E. They did, however, promote the survival of progenitors responding to the delayed addition of media conditioned by the 5637 cell line (5637 conditioned medium), rhu GM-CSF and erythropoietin. The survival promoting effects of IL-1 alpha on CFU-GM and BFU-E were neutralized by anti-IL-1 alpha mAb added to the cultures. The survival promoting effect of IL-1 alpha did not appear to be mediated by CSF, because neither CSF nor erythroid burst promoting activity were detectable in cultures in which NALT- cells were incubated with rhuIL-1 alpha. In addition, suboptimal concentrations of rhu macrophage CSF (CSF-1), G-CSF, GM-CSF, and IL-3, which were just below the levels that would stimulate colony formation, did not enhance progenitor cell survival. Survival of CFU-GM and BFU-E in low density (LD) bone marrow cells did not decrease as drastically as that in NALT- BM cells, and exogenously added IL-1 did not enhance progenitor cell survival of CFU-GM and BFU-E in LD BM cells. However, addition of anti-IL-1 beta decreased survival of CFU-GM and BFU-E in LD BM cells. These results implicate IL-1 in the prolonged survival of human CFU-GM and BFU-E.     

Differential effects of drugs upon hematopoiesis can be assessed in long-term bone marrow cultures established on nylon screens.

Hematotoxicity is associated with exposure to chemotherapeutic drugs and numerous other agents. Most measurements of the hematopoietic effects of prospective therapeutic drugs and environmental agents have been made in animal models. We tested the influence of various drugs on hematopoiesis in long-term cultures of Long-Evans rat bone marrow cells. These cultures were established on nylon screen-bone marrow stromal cell templates that were suspended in liquid medium. Previous phenotypic analyses of adherent zone cells of suspended nylon screen bone marrow cultures (NSBMC) using monoclonal antibodies and flow cytometry indicated that they maintain a multilineage character for extended periods in culture and display continuous proliferation of hematopoietic progenitors (colony-forming unit culture [CFU-C]). NSBMC of various ages were incubated for 21 hr with several concentrations of beta-D-cytosine arabinofuranoside, 5-fluorouracil, cyclophosphamide, or methotrexate. Adherent zone cells were dissociated enzymatically, phenotyped by flow cytometry, and assayed for colony-forming unit culture content. beta-D-cytosine arabinofuranoside, 5-fluorouracil, and methotrexate treatment of bone marrow cultures resulted in a dose-related diminution in colony-forming unit culture numbers in the adherent zones of NSBMC. Phenotypic analyses revealed similar trends but certain of these drugs manifested lineage specificities. Toxicity was also related to cyclophosphamide dose, but the presence of bone marrow stroma was necessary to demonstrate this effect in vitro. A subpopulation of these cells was found to metabolize ethoxyfluorescein ethyl ester to fluorescein after induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin, an effect which was quantified by flow cytometry. NSBMC may be used to ascertain lineage-specific toxicities and evaluate the effects of drugs on the proliferation of hematopoietic progenitor cells.     

The Antioxidant and Antigenotoxic Effects of Pycnogenol<Superscript>®</Superscript> on Rats Treated With Cisplatin

Oxidative stress and inflammation are implicated in the pathogenesis of cisplatin-induced toxicity. Pycnogenol® is known for its strong antioxidant and anti-inflammatory effects. In this study, the possible protective effects of pycnogenol on kidney, bone marrow, and red blood cells in rats treated with cisplatin were investigated. The rats were divided into four groups. Group 1 was the control and groups 2, 3, and 4 were orally treated with pycnogenol (200 mg/kg bw, o.p) for 5 days, treated with cisplatin (7 mg/kg bw, i.p.) on the fifth day and treated with cisplatin plus pycnogenol, respectively. Antioxidative parameters in kidney and red blood cells were measured. Chromosome anomalies in bone marrow and renal histopathology were also investigated. Activities of pro-oxidant enzymes (myeloperoxidase and xanthine oxidase), malondialdehyde, and nitric oxide levels significantly increased but antioxidant enzymes activities decreased in the kidneys and red blood cells after cisplatin treatment. Pycnogenol treatment prior to the administration of cisplatin significantly decreased cisplatin-induced injury, as evidenced by its normalizing these parameters. Chromosomal aberrations decreased and mitotic index frequencies increased in bone marrow treated with cisplatin plus pycnogenol. These findings suggest that pycnogenol may be a useful protective agent against the toxicity associated with cisplatin therapy.     

Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into multiple cell lineages. Presently, bone marrow is considered as a prime source of MSCs; however, there are some drawbacks and limitations in use of these MSCs for cell therapy. In this study, we demonstrate that human gingival tissue-derived MSCs have several advantages over bone marrow-derived MSCs. Gingival MSCs are easy to isolate, homogenous and proliferate faster than bone marrow MSCs without any growth factor. Importantly, gingival MSCs display stable morphology and do not loose MSC characteristic at higher passages. In addition, gingival MSCs maintain normal karyotype and telomerase activity in long-term cultures, and are not tumorigenic. Thus, we reveal that human gingiva is a better source of MSCs than bone marrow, and large number of functionally competent clinical grade MSCs can be generated in short duration for cell therapy in regenerative medicine and tissue engineering.     

Changes in the haematopoietic progenitor cell compartment in the acquired immunodeficiency syndrome

In order to elucidate the mechanisms responsible for the disturbances of haematopoiesis in HIV-infected individuals, bone marrow from 25 patients with either ARC or AIDS was studied. There is a stage-related decrease in CFU-GEMM, CFU-MK, BFU-E and CFU-GM, with the latter being least affected. This decrease is inversely correlated with the number of circulating CD4 cells and the CD4/CD8 ratio. Immunohistochemical and in situ hybridization studies of haematopoietic colonies failed to demonstrate HIV infection of haematopoietic cells. Neither the depletion of adherent mononuclear cells from haematopoietic cell cultures nor the addition of plasma containing antibodies against HIV gp120 could demonstrate an inhibitory effect of HIV-infected macrophages or immune-mediated progenitor cell lysis, respectively. Hence, imbalances of T-cell subpopulations appear to be mainly responsible for the progressive impairment of proliferation and differentiation of bone marrow progenitor cells observed in HIV-infected individuals.     

Platelet-rich plasma vs bone marrow aspirate concentrate: An overview of mechanisms of action and orthobiologic synergistic effects

The use of orthobiologics as a novel therapy for the treatment of numerous musculoskeletal disorders has increased considerably over the past decade.Currently,there are multiple alternatives available as suitable treatments;however,the use of autologous blood-derived products such as platelet-rich plasma(PRP),bone marrow aspirate(BMA)and BMA concentrate(BMAC),specifically,is expanding.Although many investigations attempted to demonstrate the effectiveness of these therapies,even with positive results,the literature lacks standardized protocols and overall accuracy in study designs,which leads to variance and difficulty in reproducibility of protocols.The efficacy of PRP for the treatment of cartilage,bone and muscle tissues is well known.Although BMAC has generated optimistic results for the same purposes,its applicability in clinical trials is still relatively recent when compared to PRP.Both products demonstrate the potential to set forth reparative processes,each in their own distinct mechanism.The combination of these biological products has been previously proposed,yet little is known about their synergism.Evidence indicates that growth factor,cytokine,and chemokine profiles seen in both PRP and BMAC vary but are likely to work synergistically to enhance musculoskeletal healing.BMAC products seem to work well without PRP;however,the addition of PRP to BMAC has been shown to act as a rich and natural source of culture medium for stem cells located either peripherally or in the bone marrow itself.Nevertheless,additional variables associated with the use of BMAC and PRP in orthopedics must be further evaluated in order to consolidate the efficacy of this therapeutic strategy.     

Pluripotential hematopoietic stem cells in post-5-fluorouracil murine bone marrow express the Thy-1 antigen

Expression of the Thy-1 alloantigen by hematopoietic stem and progenitor cells in post-5-fluorouracil (5-FU) murine bone marrow was investigated. FACS analysis of BDF1 bone marrow stained for Thy-1.2 with a triple-layer amplified labeling technique demonstrated that 35% of the total bone marrow population expressed Thy-1.2 (Thy-1.2+). Two distinct size subpopulations were observed in post-5-FU BDF1 marrow. Thy-1.2+ cells were present in both the large and the small subpopulations. FACS-separated bone marrow cells were also plated in methylcellulose cultures. Ninety percent of all colony-forming cells surviving in vivo administration of 5-FU were Thy-1.2+. Replating of primary hemopoietic colonies and morphologic examination of primary and secondary colonies demonstrated that the most primitive stem cells including "stem" (S) cells were Thy-1.2+. These cells (Thy-1.2+) were capable of self-renewal in vitro and exhibited multiple differentiation potentials in comparison to Thy-1.2-cells, which lacked significant self-renewal capability and were mono- or bipotent progenitor cells. Separation of Thy-1.2+ cells into large or small Thy-1.2+ subpopulations showed that only the large Thy-1.2+ colony-forming cells possessed significant self-renewal capacity. Treatment of BDF1 bone marrow with anti-Thy-1.2 plus complement reduced primary colony formation by 67% and eliminated those colony-forming cells which had extensive self-renewal properties. In the presence of PWMSCM, depletion and reconstitution of T lymphocytes had no effect on primary or secondary colony formation. These data demonstrate that Thy-1 is present on primitive hematopoietic stem cells in post-5-FU bone marrow. In addition, they show that the murine S cell is Thy-1+.     

CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo

The platelet glycoprotein IIb (alpha(IIb); CD41) constitutes the alpha subunit of a highly expressed platelet surface integrin protein. We demonstrate that CD41 serves as the earliest marker of primitive erythroid progenitor cells in the embryonic day 7 (E7.0) yolk sac and high-level expression identifies essentially all E8.25 yolk sac definitive hematopoietic progenitors. Some definitive hematopoietic progenitor cells in the fetal liver and bone marrow also express CD41. Hematopoietic stem cell competitive repopulating ability is present in CD41(dim) and CD41(lo/-) cells isolated from bone marrow and fetal liver cells, however, activity is enriched in the CD41(lo/-) cells. CD41(bright) yolk sac definitive progenitor cells co-express CD61 and bind fibrinogen, demonstrating receptor function. Thus, CD41 expression marks the onset of primitive and definitive hematopoiesis in the murine embryo and persists as a marker of some stem and progenitor cell populations in the fetal liver and adult marrow, suggesting novel roles for this integrin.     

Stem cells in liver regeneration and therapy

The liver has adapted to the inflow of ingested toxins by the evolutionary development of unique regenerative properties and responds to injury or tissue loss by the rapid division of mature cells. Proliferation of the parenchymal cells, i.e. hepatocytes and epithelial cells of the bile duct, is regulated by numerous cytokine/growth-factor-mediated pathways and is synchronised with extracellular matrix degradation and restoration of the vasculature. Resident hepatic stem/progenitor cells have also been identified in small numbers in normal liver and implicated in liver tissue repair. Their putative role in the physiology, pathophysiology and therapy of the liver, however, is not yet precisely known. Hepatic stem/progenitor cells also known as “oval cells” in rodents have been implicated in liver tissue repair, at a time when the capacity for hepatocyte and bile duct replication is exhausted or experimentally inhibited (facultative stem/progenitor cell pool). Although much more has to be learned about the role of stem/progenitor cells in the physiology and pathophysiology of the liver, experimental analysis of the therapeutic value of these cells has been initiated. Transplantation of hepatic stem/progenitor cells or in vivo pharmacological activation of the pool of hepatic stem cells may provide novel modalities for the therapy of liver diseases. In addition, extrahepatic stem cells (e.g. bone marrow cells) are being investigated for their contribution to liver regeneration. Hepatic progenitor cells derived from embryonic stem cells are included in this review, which also discusses future perspectives of stem cell-based therapies for liver diseases.     

Lymphokine-activated killer cells in rats: generation of natural killer cells and lymphokine-activated killer cells from bone marrow progenitor cells

The coculture of rat bone marrow cells with recombinant interleukin-2 induced the generation of cells mediating natural killer (NK) activity and subsequent lymphokine-activated killer (LAK) activity depending upon the dose of IL-2 and time of culture. NK activity was detected as early as 4 to 5 days after the addition of IL-2 and could be evoked with as little as 5 to 50 U/ml. The induced NK cells had large granular lymphocyte (LGL) morphology and expressed 0X8 and asialo GM1 surface markers but did not express 0X19 or W3/25 markers. LAK activity was detected only after 5 days of culture, and required above 100 U/ml IL-2. Cells mediating LAK activity also expressed 0X8 and asialo GM1 but not 0X19. The generation of detectable NK and subsequent LAK activity was due to induction of early progenitor cells and not contaminating mature LGL/NK cells within the bone marrow population since of removal of such mature NK cells with L-leucine methyl ester (L-LME) did not affect the subsequent generation of either activity. Moreover, the removal of actively dividing cells as well as mature NK cells from the bone marrow by treatment with 5-fluorouracil (5-FU) in vivo enriched the remaining bone marrow population for both NK and LAK progenitor cells. The phenotype of the L-LME- and 5-FU-resistant NK and LAK progenitor cells within populations of bone marrow was determined by antibody plus complement depletion analysis. Although treatment of normal bone marrow with anti-asialo GM1 + C reduced the induction of NK and LAK activity in 5-day cultures, treatment of 5-FU marrow with anti-asialo GM1 + C did not affect either activity. Treatment with a pan-T cell antibody + C did not affect the development of NK or LAK activity under any conditions. Thus, the 5-FU-resistant NK/LAK progenitors were asialo GM1 negative but became asialo GM1+ after induction by IL-2. Finally, evidence that bone marrow-derived LAK cells were generated directly from the IL-2-induced NK cells was obtained by treating the IL-2-induced LGL/NK cells with L-LME.(ABSTRACT TRUNCATED AT 400 WORDS)     

Coadministration of swainsonine and doxorubicin attenuates doxorubicin-induced lethality in mice.

This study in mice concerns the protective effectiveness and mechanisms of action by which a coadministered regimen of an immunomodulatory alkaloid swainsonine (8alphabeta-indolizidine-1alpha,2alpha,8beta-triol) protects against lethality induced by a single bolus intraperitoneal injection of LD50/14 doxorubicin. This swainsonine coadministration treatment regimen has been identified previously in our laboratory as the superior of the two optimal conditions for diminishing lethality in mice due to LD50/14 doxorubicin. The anthracycline, doxorubicin is a potent and widely used cancer chemotherapeutic agent whose clinical usefulness is limited by both a dose- and time-dependent cardiomyopathy. Specifically, mice were given simultaneous injections of swainsonine or its diluent buffer, phosphate buffered saline and LD50/14 doxorubicin on day 0, followed by twice daily injections of swainsonine or phosphate buffered saline up to day +9. The survival and well being of mice were monitored daily for 70 days, which may be considered equivalent to a period of 4 to 5 years in humans. This duration has a clinical implication with respect to the late manifestation of cardiotoxicity after doxorubicin treatment. We quantified the bone marrow cellularity of mice and performed in vitro progenitor cell assays to determine the effects of swainsonine coadministration treatment regimen on bone marrow competence after doxorubicin treatment. The effects of this regimen on doxorubicin-induced changes in heart morphology and on hematologic toxicities caused by doxorubicin were determined. This swainsonine coadministration treatment regimen significantly diminished doxorubicin-induced lethality and prolonged survival and well being of mice by preventing bone marrow pancytopenia from the start of therapy. It decreased bone marrow toxicity and facilitated its restoration. It accelerated restoration of blood hematocrit and total leukocyte levels. Also it facilitated the proliferation and differentiation of bone marrow pluripotent stem cells along the different paths to progenitor lineages, and significantly preserved the mouse heart morphology. These underlying mechanisms of action for the protection by swainsonine coadministration strongly suggest a potential role for swainsonine in high dose chemotherapy with doxorubicin.     

Regulation of natural killer progenitors. Studies with a novel immunomodulator with distinct effects at the precursor level

NK cells originate from progenitors in the bone marrow and maturate independently of other lymphoid organs. NK cell maturation represents an important site for regulation of the level of NK activity and constitutes a potentially interesting target for therapeutic intervention. The effect of the immunomodulator Linomide (carboxamide-3-quinoline) on the regeneration of NK cells was studied in vivo after depletion of mature NK cells. Linomide significantly, although to various extent, accelerated the maturation of NK cells after specific depletion with antibodies to asialomonoganglioside, treatment with cyclophosphamide or lethal irradiation and syngeneic bone marrow grafting. Examination of the target cell spectrum lysed by spleen effector cells during Linomide treatment as well as studies of phenotype, clearly indicated that the effector cells studied were NK cells. Treatment of mice for 4 days with Linomide increased the frequency of bone marrow NK cell progenitors from 1/11,900 to 1/6,000 as judged by limiting dilution analysis. Direct addition of Linomide in vitro had no effect on cultures of mature NK cells from spleen, but had an additive effect to IL-2 on the generation of NK cells when added to bone marrow cultures. Our study indicates that different mechanisms exist for the regulation of progenitor and mature NK cells, and that the immunomodulator Linomide represent a potentially important tool for investigating the mechanisms governing NK cell maturation.