Biomedical subjects

A Tsukamoto

Publications and source records attributed to A Tsukamoto.

At least 19 recordsLinked to original sources

PubMed2000

[Autoimmune hepatitis complicated by intolerable pain of lower extremities and shock due to azathioprine].

A 24-year-old woman was followed for about ten months with oral administration of prednisolone (22.5-35 mg/d) for autoimmune hepatitis. In June 1995, she noticed fatigue and appetite loss and blood chemistry revealed markedly deteriorated liver function. She was admitted to our hospital. The daily dose of prednisolone was increased to 60 mg. Her elevated levels of transaminases decreased gradually. Administration of azathioprine (100 mg/d) was started with tapering of prednisolone on August 18th. Ten days later, tender cervical lymphadenopathy and high fever occurred. Azathioprine administration was stopped immediately and intravenous antibiotics were given. On September 5th, 50 mg of azathioprine was administered again. Two hours later, the patient complained of intolerable pain from the lumbar region to the knee joints, which subsided following two injections of analgesics within a few hours. However, chills, high fever and hypotension (86/30 mmHg) subsequently developed. No bacterial growth was detected in blood culture. She was discharged on September 12th. On October 4th, she visited our out-patient clinic. The next day, she took one tablet (50 mg) of azathioprine at 10 o'clock. She noted intense pain from the thighs to the knees and calves around noon again. Her home doctor found that she exhibited shock (BP 67/?). She was immediately taken to our department. The same symptoms and signs as the above-mentioned occurred. Azathioprine was considered responsible for these two adverse reactions (shock) as an allergen. Later, systemic lupus eythematosus was diagnosed in 1996. And she died to pulmonary hypertension in May 1999. Physicians should be aware of the potential adverse effect of azathioprine administered in order to manage the patients with autoimmune disorders.

Adult↗

PubMed1999

Regulation of angiogenesis in human hepatomas: possible involvement of p53-inducible inhibitor of vascular endothelial cell proliferation.

Mechanisms regulating angiogenesis in human hepatomas were analyzed. Huh7 hepatoma cells transplanted into athymic mice formed highly vascularized reddish tumors with abundant microvessels, while angiogenesis by PLC/PRF/5 hepatoma cells was less remarkable. However, the production of angiogenesis stimulators such as VEGF and IL6 by Huh7 cells was much less than by PLC/PRF/5 cells. In addition, the production of angiogenesis inhibitor TSP-1 by Huh7 cells was greater than by PLC/PRF/5 cells. Therefore, the difference in angiogenesis between these two hepatomas was not explained by the production of these known angiogenesis regulators. On the other hand, PLC/PRF/5 cells but not Huh7 cells secreted an inhibitor of the proliferation of vascular endothelial cells, which was enhanced by p53-gene transfer. These results suggest that the production of this p53-inducible angiogenesis inhibitor is responsible, at least partly, for the regulation of angiogenesis in human hepatomas.

Animals↗

PubMed1999

[Adult T-cell leukemia with elevated serum hyaluronic acid levels paralleling disease activity].

We report on two adult T-cell leukemia (ATL) patients whose levels of serum hyaluronic acid (HA) moved in parallel with the clinical activity of their disease. A 66-year-old man was admitted to our hospital because of unconsciousness and hypotension. Acute type ATL complicated by hypercalcemia and myelofibrosis was diagnosed. Before therapy, the level of patient's serum HA was 2,045 to 4,010 ng/ml (normal range: 50 >). After he achieved complete remission (CR) through chemotherapy, his serum HA was 36 ng/ml. Several months later, however, his ATL relapsed, and his serum HA increased to 393 ng/ml. The other patient was an 80-year-old man who had been admitted on the suspected diagnosis of ATL. Before chemotherapy, his serum HA was high (3,420 ng/ml). After CHOP therapy, he entered CR and his HA decreased to 122 ng/ml. He remains in CR with slightly elevated levels of HA (127 to 212 ng/ml), and is being followed up on an out-patient basis.

Aged↗

PubMed1998

Suppression of hepatoma growth and angiogenesis by a fumagillin derivative TNP470: possible involvement of nitric oxide synthase.

TNP470, a derivative of fumagillin, suppressed in vivo growth of human PLC/PRF/5 hepatoma and ameliorated cachexia of hepatoma-bearing mice. These in vivo effects were associated with reductions in microvessel and macrophage counts. In in vitro experiments, TNP470 inhibited the growth and migration of human hepatoma and bovine vascular endothelial (VE) cells. TNP470 did not inhibit the production of VE growth factor by the hepatoma, which suggests that this compound acts directly on VE cells in vivo. In contrast, TNP470 inhibited the production of leukemia inhibitory factor, which may be related to the amelioration of cancer cachexia. TNP470 induced apoptosis and enhanced the expression of beta-galactosidase, a biomarker of senescence, which was partly mimicked by a nitric oxide (NO) donor S-nitroso-N-acetyl penicillamin. TNP470 inhibited myristoylation and membrane translocation of NO synthase and increased the cellular content of NO synthase and production of NO. Therefore, it is suggested that the actions of TNP470 are mediated, at least in part, through the inhibition of membrane translocation of biologically active proteins.

Animals↗

PubMed1998

2-Methoxyestradiol, an endogenous metabolite of estrogen, enhances apoptosis and beta-galactosidase expression in vascular endothelial cells.

2-Methoxyestradiol (2ME) is an endogenous metabolite of estradiol (E2) and is known to inhibit tumor angiogenesis. In the present study, the direct effects of 2ME on the vascular endothelial cells were examined. 2ME enhanced apoptosis and beta-galactosidase expression in bovine vascular endothelial cells. A nitric oxide (NO) donor S-nitroso-N-acetyl penicillamin (SNAP) also enhanced beta-galactosidase expression, suggesting a possible role of NO in mediating the action of 2ME. 2ME increased the cellular content of nitric oxide synthase (NOS) and the production of NO. In addition, 2ME altered the membrane localization pattern of NOS. These suggest that the effects of 2ME on apoptosis and senescence of vascular endothelial cells were mediated, at least partly, by NOS and NO.

2-Methoxyestradiol↗

PubMed1998

Collection, tumor contamination, and engraftment kinetics of highly purified hematopoietic progenitor cells to support high dose therapy in multiple myeloma.

Unfractionated peripheral blood stem cell (PBSC) grafts contain measurable quantities of myeloma cells and are therefore a potential source of relapse posttransplantation. In contrast, fluorescence-activated cell sorting (FACS)-sorted CD34+ Thy1+ Lin- peripheral blood cells are substantially enriched for stem cell activity, yet contain virtually no clonal myeloma cells. A study was performed in patients with symptomatic myeloma, who had received 12 months or less of preceding standard chemotherapy, to evaluate the feasibility of large scale purification of primitive hematopoietic stem cells in order to study engraftment kinetics posttransplantation and the degree of tumor cell contamination of this cell population, based on polymerase chain reaction (PCR) analysis for the patient-specific complementarity-determining region III (CDR III). PBSC were mobilized with high dose cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF). A combination of elutriation and chemical lysis was used to deplete PBSC collections of monocytes, granulocytes, erythrocytes, and platelets. Subsequently, CD34+ Thy1+ Lin- progenitor cells were purified with high speed cell sorting. Of the 10 evaluable patients, nine met the required minimum criteria of >/=7.2 x 10(5) cells/kg to support tandem transplants. After high dose melphalan (200 mg/m2) eight engrafted successfully, although granulocyte (absolute neutrophil count [ANC] >0.5 x 10(9)/L, 16 days) and platelet recovery (platelets > 50 x 10(9)/L, 39 days) was substantially delayed when compared with unmanipulated PBSC grafts; one patient required infusion of a reserve graft because of lack of evidence of engraftment by day +28. Three patients proceeded to a second graft with high dose melphalan and total body irradiation; two required infusion of a reserve graft and both died of infectious complications; one showed delayed, but complete, engraftment after this myeloablative regimen. Two of the nine evaluable patients attained a clinical complete remission (CR). The grafts from three patients were tested for tumor contamination and contained no detectable clonal myeloma cells. Larger quantities of purified cells may be required to resolve the problem of delayed engraftment.

Adult↗

PubMed1998

High doses of purified stem cells cause early hematopoietic recovery in syngeneic and allogeneic hosts.

In humans, autologous transplants derived from bone marrow (BM) usually engraft more slowly than transplants derived from mobilized peripheral blood. Allogeneic BM transplants show a further delay in engraftment and have an apparent requirement for donor T cells to facilitate engraftment. In mice, Thy-1.1(lo)Lin-/loSca-1+ hematopoietic stem cells (HSCs) are the principal population in BM which is responsible for engraftment in syngeneic hosts at radioprotective doses, and higher doses of HSCs can radioprotect an allogeneic host in the absence of donor T cells. Using the mouse as a preclinical model, we wished to test to what extent engraftment kinetics was a function of HSC content, and whether at high doses of c-Kit+Thy-1.1(lo)Lin-/loSca-1+ (KTLS) cells rapid allogeneic engraftment could also be achieved. Here we demonstrate that engraftment kinetics varied greatly over the range of KTLS doses tested (100-10,000 cells), with the most rapid engraftment being obtained with a dose of 5,000 or more syngeneic cells. Mobilized splenic KTLS cells and the rhodamine 123(lo) subset of KTLS cells were also able to engraft rapidly. Higher doses of allogeneic cells were needed to produce equivalent engraftment kinetics. This suggests that in mice even fully allogeneic barriers can be traversed with high doses of HSCs, and that in humans it may be possible to obtain rapid engraftment in an allogeneic context with clinically achievable doses of purified HSCs.

Animals↗

PubMed1998

Development of a simple high-performance capillary electrophoretic method with on-line mode in capillary derivatization for the determination of spermidine.

A new high-performance capillary electrophoretic (HPCE) method with an on-line mode in-capillary derivatization (ICD) procedure for determinations of some amines using 20 mmol/L sodium dodecyl sulfate (SDS) - 2 mmol/L o-phthalaldehyde (OPA) - 2 mmol/L N-acetylcysteine (NAC) - 20 mmol/L phosphate-borate buffer [9] has previously been shown. Although this technique offers direct fluorescence detection of free amines without any derivatization procedures before or after HPCE separation, the presence of spermidine (Spd) is difficult to detect due to low fluorescence intensity. The purpose of this study is to improve the detection sensitivity of Spd by reoptimizing this method with regard to the run buffer; the reoptimized method was applied to the determination of Spd in human plasma. To enhance the fluorescence intensity of the Spd signal, it is effective to use the run buffer in the presence of both beta-cyclodextrin (beta-CD: 8.8 mmol/L) and NAC at high concentration (16 mmol/L). By contrast, the intensity was remarkably decreased when SDS was used in the presence of beta-CD. After ultrafiltrating (UF) spiked human plasma with Spd, UF plasma was directly analyzed using the reoptimized method. Spd peak was detected and separated from the other peaks of blank plasma. The present method gave good linearity (r = 0.999), reproducibility (3.85% coefficient of variation at 5 micromol/L level; n = 10) and specificity. The detection limit and lower limit of quantitation is for 0.2 micromol/L and 1 micromol/L, respectively.

Buffers↗

PubMed1998

Cystothiazoles A and B, new bithiazole-type antibiotics from the myxobacterium Cystobacter fuscus.

New bithiazole-type antibiotics, cystothiazoles A (C20H26N2O4S2) and B (C20H26N2O5S2), have been isolated from a culture broth of the myxobacterium, Cystobacter fuscus. The gross structures of these compounds were elucidated by spectroscopic analysis, and their absolute stereochemistry was determined by chemical degradation of cystothiazole A. Cystothiazole A inhibits fungi and human tumor cells, whereas it is inactive against bacteria. The antifungal activity appears to result from the inhibition of submitochondrial NADH oxidation. Although these compounds are structurally related to the known antibiotic myxothiazol, cystothiazole A was more active against fungi and less cytotoxic than myxothiazol.

Anti-Bacterial Agents↗

PubMed1998

[Allogeneic peripheral blood stem cell transplantation in 30 patients with hematologic disorders].

Thirty patients (median age of 32 years; range, 6-61) with hematologic disorders received unmanipulated peripheral blood stem cell transplants from HLA-matched or one-antigen-mismatched related donors following myeloablative therapy for acute lymphoblastic leukemia (7), acute myelogenous leukemia (6), chronic myelogenous leukemia (8), myelodysplastic syndrome (3), or other disorders (6). Granulocyte colony stimulating factor (G-CSF) mobilized peripheral blood stem cells were collected from donors in 1 to 3 aphereses. The apheresis products contained mean counts of 11.3 x 10(8) (range, 3.8-17.2) nucleated cells/kg and 6.7 x 10(6) (range, 1.3-16.7) CD34+ cells/kg. Graft-versus-host-disease (GVHD) prophylaxis consisted of cyclosporin A plus methotrexate, or FK506 plus methotrexate. All patients received G-CSF following their transplant. Although 1 patient died of pneumonia 6 days after transplantation, the others demonstrated rapid engraftment. Median days to recovery to 500/microliter neutrophils and 20,000/microliter platelets were 13 (range, 8-21) and 14 (range, 1-23) days, respectively. The incidence of acute GVHD grade II-IV was 33%; chronic GVHD developed in 57% of the assessable patients. There were no episodes of graft failure or rejection. Nineteen patients (63%) were alive and in complete remission from 147 to 839 days following their transplant (median follow-up of 560 days). Further follow-up study will be required to assess the incidence of chronic GVHD and graft-versus-leukemia (GVL) effects.

Adolescent↗

PubMed1998

Bifid ribs observed in the third and the fourth ribs.

Three cases of bifid ribs were found in two cadavers during routine dissections at the Iwate Medical University School of Dentistry. All of the cases were found in the third or the fourth rib. The distal parts of the osseous rib bifurcated with an angle of 60 degrees and both of the branches had their own costal cartilage. The costal cartilage fused again to form the trunk which was connected to the sternum. The space between the two branches was filled with presumably normal intercostal muscles. Blood supply was maintained by a small branch from the interthoracic artery to the upper branches. However, the intercostal nerves did not branch toward the upper branch but only ran along the lower margins of the lower branches of the bifid ribs.

Aged↗

PubMed1997

The unexpected G0/G1 cell cycle status of mobilized hematopoietic stem cells from peripheral blood.

Treatment with a combination of cytokines and chemotherapy can effectively stimulate the release of hematopoietic stem cells (HSC) into the peripheral blood (PB), which can then be harvested for transplantation. The cell cycle status of the harvested HSC from mobilized PB (MPB) is of interest because of the impact that cell cycling may have on optimizing the conditions for ex vivo expansion, retrovirus-mediated gene transfer, and the engraftment of transplanted tissues. Therefore, we characterized the cell cycling status of mobilized HSC from mice and humans. The murine HSC, which express the phenotype c-kit+ Thy-1.1lo Lin-/lo Sca-1+, were purified from PB, bone marrow (BM), and spleen after the mice were treated with the mobilizing regimen of granulocyte colony-stimulating factor (G-CSF) or a combination of cyclophosphamide (CTX) and G-CSF. Human HSC (CD34+ Thy-1+ Lin-) and progenitor cells (CD34+ Thy-1-Lin-) were isolated from the BM of untreated healthy volunteers and from MPB of healthy volunteers and patients treated with G-CSF or a combination of CTX and GM-CSF. Cell cycle status was determined by quantitating the amount of DNA in the purified cells after staining with the dye Hoechst 33342. Fluorescence-activated cell sorting analysis of the progenitor cells from the murine and human samples showed an unexpected finding, ie, virtually none of the cells from the MPB was cycling. The G0/G1 status of HSC from MPB was surprising, because a significant proportion of HSC from BM are actively proliferating and, after mobilization, the HSC in the spleen and BM were also actively cycling.

Animals↗

PubMed1997

[Disseminated intravascular coagulation in a case of adult onset Still's disease].

We report a 82-year-old woman with adult onset Still's disease (AOSD), who presented with high fever, skin rash, swollen axillary lymph nodes, accelerated erythrocyte sedimentation rate, leukocytosis, abnormal liver function tests, hypoalbuminemia, negative antinuclear antibody and rheumatoid factor, and lack of renal involvement. Disseminated intravascular coagulation (DIC) was also diagnosed on admission. An antipyretic relieved high fever and DIC soon improved. Three years later, AOSD relapsed accompanied by hypercoagulation and hyperfibrinolysis. The patient developed subdural hematoma and DIC due to a brain contusion. High titers of serum soluble adhesion molecules and soluble thrombomodulin were noted on the first episode of DIC. These findings indicated that endothelial cells were damaged in AOSD complicated by DIC.

Aged↗

PubMed1996

Partially differentiated ex vivo expanded cells accelerate hematologic recovery in myeloablated mice transplanted with highly enriched long-term repopulating stem cells.

The ability of an infusion of ex vivo expanded hematopoietic cells to ameliorate cytopenia following transplantation of hematopoietic stem cells (HSCs) is controversial. To address this issue, we measured the recovery of circulating leukocytes, erythrocytes, and platelets in lethally irradiated mice transplanted with 10(3) enriched HSCs, with or without their expanded equivalent (EE) generated after 7 days of culture in interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor and Steel Factor. Two HSC populations differing in their content of short-term repopulating progenitors were evaluated. Thy-1loLIN-Sca-1+ (TLS) bone marrow (BM) is enriched in colony-forming cells (CFCs), day 8 and day 12 spleen colony-forming units (CFU-S) (435 +/- 19, 170 +/- 30, and 740 +/- 70 per 10(3) cells, respectively), and stem cells with competitive long-term repopulating potential (> or = 1 per 43 cells). Thy-1loSca-1+H-2Khl cells (TSHFU) isolated from BM 1 day after treatment of donor mice with 5-fluorouracil (5-FU) are also highly enriched in competitive repopulating units (CRU, > or = 1 per 55 cells), but are depleted of CFCs, day 8 and day 12 CFU-S (171 +/- 8, 0 and 15 +/- 4 per 10(3) cells, respectively). Recipients of 10(3) TLS cells transiently recovered leukocytes to > or = 2,000/microL in 12 days, but sustained engraftment required 25 days. Platelets recovered to > or = 200,000/microL in 15 days, and erythrocytes never decreased below 50% of normal. Mice transplanted with 10(3) TSHFU cells recovered leukocytes in 15 days, and platelets and erythrocytes in 18 days. Recipients of unseparated normal or 5-FU-treated BM cells (containing 10(3) TLS or TSHFU cells) recovered safe levels of blood cells in 9 to 12 days, suggesting that unseparated marrow contains early engrafting cells that were depleted by sorting. Upon ex vivo expansion, total cells, CFCs and day 12 CFU-S were amplified 2,062-,83- and 13-fold, respectively, from TLS cells; and 1,279-, 259- and 708-fold, respectively, from TSHFU cells. Expanded cells could regenerate the majority of lymphocytes and granulocytes in primary (17 weeks) and secondary (26 weeks) hosts and were only moderately impaired compared to fresh HSCs. The EE of TSHFU cells was more potent than that of TLS cells, suggesting that more highly enriched HSCs are more desirable starting populations for this application. When mice were transplanted with 10(3) TSHFU cells and their EE, the duration of thrombocytopenia was shortened from 18 to 12 days, and anemia was abolished. Leukocytes were also elevated on days 9 to 12, although sustained recovery was not accelerated. Anemia was also abrogated in recipients of 10(3) TLS cells and their EE. Early platelet counts were slightly higher than with TLS cells alone, but leukocyte recovery was not improved. These data confirm that TLS cells contribute to early and sustained hematopoiesis, and demonstrate a benefit of ex vivo expanded cells in accelerating engraftment of more primitive TSHFU stem cells depleted of progenitors.

Animals↗

PubMed1996

Structural characteristics of cationic liposomes with potent enhancing effect on retroviral transduction into human hepatoma cells.

To improve the efficiency of retroviral transduction into human hepatoma cells, new liposomes were prepared using different cationic and neutral lipids. Their effect on the retroviral transduction was evaluated using PLC/PRF/5 hepatoma cells and Moloney murine leukemia virus-derived retrovirus carrying herpes simplex thymidine kinase gene. Those liposomes consisted of cationic lipids with a long spacer were highly efficient in enhancing retroviral transduction and also less cytotoxic. On the other hand, the length of hydrophobic domains of neutral lipids was not correlated with the efficiency in enhancing the retroviral transduction. These results suggest that liposomes which effectively enhance retrovirus transduction can be developed by using cationic lipids with new spacers.

Carcinoma, Hepatocellular↗

PubMed1996

Primitive human hematopoietic cells displaying differential efflux of the rhodamine 123 dye have distinct biological activities.

Human bone marrow (BM) CD34+ cells were stained with the vital dye, rhodamine 123 (Rh123), and analyzed for their biological properties based on the level of dye retention. Heterogeneous rhodamine staining is seen within the CD34+ population, and the staining patterns differ dramatically between fetal BM (FBM), adult BM (ABM) and mobilized peripheral blood (MPB). Kinetic analysis of the efflux of Rh123 from ABM CD34+ cells showed that efflux of Rh123 was most rapid from the most primitive Thy-1+ subset. The efflux of Rh123 could be inhibited by verapamil, suggesting that rhodamine efflux from primitive hematopoietic cells is primarily due to the P-glycoprotein (P-gp) pump or another intracellular transport system affected by verapamil. When four CD34+ subpopulations were plated onto SyS1 BM stromal cell cocultures after 1 to 2 weeks, only wells plated with CD34+ Thy-1+Rh123lo (low-level Rh123 retention) or CD34+Thy-1+Rh123mid (mid-level Rh123 retention) cells maintained greater than 50% of cells in an uncommitted CD34+33- stage. CD34+Lin- (lineage-negative) cells were fractionated based on Rh123 dye staining into Rh123hi (high-level Rh123 retention), Rh123mid, and Rh123lo and deposited as single cells into long-term SyS1 BM stromal cell cultures. The Rh123mid fraction had immense early proliferative activity in vitro, but lost the ability to form cobblestone areas after 5 to 6 weeks in culture. In contrast, the Rh123lo fraction proliferated more slowly but sustained long-term in vitro hematopoiesis as evidenced by continued cobblestone area-forming cells (CAFC) activity for at least 6 weeks. The Rh123hi fraction showed a plating efficiency similar to that of the Rh123lo or Rh1123mid fractions but did not extensively proliferative in vitro and did not show evidence of CAFC activity. We predicted from these in vitro results that the Rh123lo subsets possesses long-term engrafting potential. Indeed, on transplantation into the SCID-hu bone assay, all long-term engrafting potential and multilineage differentiation potential resided within the Rh123lo-mid but not Rh123hi subset. Furthermore, human marrow subpopulations derived from chimeric sheep after in utero transplantation with CD34+Thy-1+Lin- cells were reisolated based on Rh123 staining. Again, CD34+Lin- subsets showing Rh123lo-mid had long-term growth in culture, whereas Rh123hiCD34+Lin- cells did not. These results show that, after injection of CD34+Thy-1+Lin- cells into an in utero microenvironment, primitive CD34+ cells maintain a Rh123 phenotype that correlates with their in vitro CAFC activity. Thus, Rh123 staining is an effective way to define functional subsets of primitive hematopoietic cell populations.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Cationic liposomes enhance retrovirus-mediated multinucleated cell formation and retroviral transduction.

A retroviral vector carrying herpes simplex virus thymidine kinase (HSV-TK) gene was introduced into psi 2 packaging cells (psi 2tkn) and XC tumor cells (XCtkn). psi 2tkn, XCtkn and XC cells co-cultured with TK-carrying cells were killed by ganciclovir (GCV) treatment. The growth of XC cells transplanted into nude mice was also suppressed by intratumoral injection of psi 2tkn or XCtkn cells and subsequent GCV administration. In addition, the XC cells cultured with either psi 2tkn cells or cell-free retrovirus suspension formed giant multinucleated cells. The multinucleated cell formation was specific to the combination of XC cells and the retrovirus produced by the psi 2tkn cells. Cationic liposomes enhanced the retrovirus-induced multinucleated cell formation and retroviral transduction. The correlation between the two actions of liposomes suggests that liposomes which enhance multinucleated cell formation are potent enhancers of retroviral transduction.

Animals↗

PubMed1996

Leukemia inhibitory factor upregulates cytokine expression by a murine stromal cell line enabling the maintenance of highly enriched competitive repopulating stem cells.

Attempts to maintain or expand primitive hematopoietic stem cells in vitro without the concomitant loss of their differentiative and proliferative potential in vivo have largely been unsuccessful. To investigate this problem, we compared the ability of three cloned bone marrow (BM) stromal cell lines to support the growth of primitive Thy-1lo Sca-1+H-2Khi cells isolated by fluorescence-activated cell sorting from the BM of Ly-5.2 mice treated 1 day previously with 5-fluo- rouracil. Sorted cells were highly enriched in cobblestone area-forming cells (CAFC), but their frequency was dependent on the stromal cell lines used in this assay (1 per 45 cells on SyS-1; 1 per 97 cells on PA6). In the presence of recombinant leukemia inhibitory factor (LIF), CAFC cloning efficiency was increased to 1 per 8 cells on SyS-1 and 1 per 11 cells on PA6, thus showing the high clonogenicity of this primitive stem cell population. More primitive stem cells with competitive repopulating potential were measured by injecting the sorted cells into lethally irradiated Ly-5.1 mice together with 10(5) radioprotective Ly-5.1 BM cells whose long-term repopulating ability has been "compromised" by two previous cycles of marrow transplantation and regeneration. Donor-derived lymphocytes and granulocytes were detected in 66% of animals injected with 50 sorted cells. To quantitate the maintenance of competitive repopulating units (CRU) by stromal cells, sorted cells were transplanted at limiting dilution before and after being cultured for 2 weeks on adherent layers of SyS-1, PA6, or S17 cells. CRU represented 1 per 55 freshly sorted cells. CRU could be recovered from cocultures supported by all three stromal cell lines, but their numbers were approximately-sevenfold less than on day 0. In contrast, the addition of LIF to stromal cultures improved CRU survival by 2.5-fold on S17 and PA6 cells (approximately two-fold to threefold decline), and enabled their maintenance on SyS-1. LIF appeared to act indirectly, because alone it did not support the proliferation of Thy-1lo Sca-1+H-2Khi cells in stroma-free cultures. Polymerase chain reaction (RT-PCR) analysis revealed that Interleukin-1beta (IL-1 beta) IL-2, IL-6, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, transforming growth factors, LIF, and Steel Factor (SLF) mRNAs were upregulated in SyS-1 within 1 to 6 hours of LIF-stimulation. To determine if increased expression of SLF by LIF-stimulated SyS-1 cells could account for their capacity to support stem cells, sorted calls were cocultured on simian CV-E cells that were transfected with an expression vector encoding membrane-bound SLF, or supplemented with soluble SLF. In both cases, SLF synergized with IL-6 produced endogenously by CV-E cells enabling CAFC growth equivalent to that on LIF-stimulated SyS-1. CAFC development on LIF-stimulated SyS-1 could also be completely abrogated by an anti-SLF antibody. These data provide evidence for a role of LIF in the support of long-term repopulating stem cells by indirectly promoting cytokine expression by BM stroma. Furthermore, we have used quantitative assays to show a maintenance of CRU numbers, with retention of in vivo function following ex vivo culture.

Animals↗