Biomedical subjects

S Li

Publications and source records attributed to S Li.

At least 37 records · Page 2Linked to original sources

PubMed2006

CTF determination and correction in electron cryotomography.

Electron cryotomography (cryoET) has the potential to elucidate the structure of complex biological specimens at molecular resolution but technical and computational improvements are still needed. This work addresses the determination and correction of the contrast transfer function (CTF) of the electron microscope in cryoET. Our approach to CTF detection and defocus determination depends on strip-based periodogram averaging, extended throughout the tilt series to overcome the low contrast conditions found in cryoET. A method for CTF correction that deals with the defocus gradient in images of tilted specimens is also proposed. These approaches to CTF determination and correction have been applied here to several examples of cryoET of pleomorphic specimens and of single particles. CTF correction is essential for improving the resolution, particularly in those studies that combine cryoET with single particle averaging techniques.

Cryoelectron Microscopy↗

PubMed2006

Influence of lipoprotein lipase gene Ser447Stop and beta1-adrenergic receptor gene Arg389Gly polymorphisms and their interaction on obesity from childhood to adulthood: the Bogalusa Heart Study.

OBJECTIVE: To investigate the influence of lipoprotein lipase (LPL) Ser447Stop and beta1-adrenergic receptor (ADRB1) Arg389Gly gene polymorphisms, individually and in combination, on obesity from childhood to adulthood. DESIGN AND SUBJECTS: A community-based cohort of 1331 subjects (30% black and 70% white subjects) was followed over an average period of 23 years from childhood (age range: 4-17 years) to adulthood (age range:18-44 years). MEASUREMENT: Body mass index (BMI, kg/m2) and LPL Ser447Stop and the ADRB1 Arg389Gly genotypes. RESULTS: The frequency of the ADRB1 Gly389 allele was 0.25 in white subjects vs 0.39 in black subjects (P < 0.001); 0.08 vs 0.05 (P = 0.280) for the LPL Stop447 allele. There was no association between the LPL Stop447 allele and BMI among white and black subjects either in childhood and adulthood levels or annual change from childhood to adulthood. The ADRB1 Gly389 allele was associated with lower BMI only in black adults (P = 0.017). Further, the interaction effect of the LPL Stop447 allele and ADRB1 Gly389 allele on adult BMI or its annual change was significant in white subjects and in the total sample (P = 0.03-0.006). Childhood values tended to show a similar trend. Having both ADRB1 Gly389 allele and LPL Stop447 allele was associated with 71% (95% confidence interval: 26-89%) less odds for developing obesity from childhood to adulthood after adjusting for age, race, sex, and childhood BMI. CONCLUSION: While Gly389 allele of the ADRB1 gene lowers obesity in black subjects, this allele in conjunction with Stop447 allele of the LPL gene lowers obesity in adults and attenuates the development of obesity from childhood to adulthood. These findings underscore the importance of gene-gene interaction in the assessment of genetic influences on complex traits such as obesity.

Adolescent↗

PubMed2006

Effect of dendritic cell vaccine against a tongue squamous cell cancer cell line (Tca8113) in vivo and in vitro.

Dendritic cells (DCs), as primary antigen-presenting cells with the capacity to activate naïve T lymphocytes, are considered to be promising adjuvants for immunity against cancer. In this study, the effect of T lymphocyte-mediated immunity induced by a DC vaccine against Tca8113 cells in vivo and in vitro was evaluated. DCs were from human peripheral blood monocytes cultured in the presence of granulocyte-macrophage colony stimulating factor, interleukin 4 and/or tumour necrosis factor alpha, and stimulated with Tca8113 cell lysate prepared by the freeze-thaw method. The autologous T cells activated by these DCs were used in an in vitro MTT assay to detect their tumouricidal activity and investigated for their anti-tumour effect in vivo by administration to nude mice with implanted tumours (Tca8113). An adequate number of DCs were successfully generated from the monocytes. The T cells activated by the DC-based vaccine killed Tca8113 cells in vitro (P<0.01), postponed tumour doubling time of the implanted tumours in nude mice (P<0.01) and inhibited the growth of the tumours (P<0.05). These results show that DCs from monocytes induce a lymphocyte-mediated immune response against tongue squamous carcinoma, and could be used as a vehicle for tumour antigens.

Animals↗

PubMed2006

Preclinical assessment of the feasibility of applying controlled release oral drug delivery to a lead series of atypical antipsychotics.

In this paper, we present a preclinical approach for evaluating the feasibility of applying controlled-release (CR) oral drug delivery to increase the duration of exposure and lower the C(max) of compounds in a lead series of short half-life atypical antipsychotics. Three lead compounds in the series had demonstrated potential pharmacological benefits for the treatment of psychosis, in preclinical studies. However, the compounds showed evidence of insufficient half-lives to enable a once-a-day (QD) product using immediate-release (IR) oral delivery. To evaluate and compare the potential for oral CR delivery to extend the duration of action and thereby enable QD administration, the in vitro solubility and permeability, and the duodenal and colonic absorption of three compounds in the series were measured. Based on the results, one candidate was selected for advancement that showed moderate in vitro solubility, but had the highest in vitro permeability and ratio of colonic to duodenal bioavailability (0.9) in the rat. The results from this study provided evidence that a CR drug delivery system could be used to extend the duration of exposure of the compounds in the series and a scientific basis for selecting one of the three compounds as a candidate.

Administration, Oral↗

PubMed2006

Unilateral spatial neglect and memory deficit associated with abnormal beta-amyloid precursor protein accumulation after lateral fluid percussion injury in Mongolian gerbils.

The purpose of this study was to investigate cognitive/memory dysfunctions and the pathological process contributing to such dysfunction following moderate lateral fluid percussion injury (LFPI) in Mongolian gerbils. Mongolian gerbils were subjected to moderate LFPI (1.3 1.6 atm). During 7 days post-trauma, spatial cognitive and memory dysfunctions were evaluated by T-maze test (TMT). At 6 hours, 24 hours, and 7 days post-injury, animals were sacrificed and the brains were prepared for Kluver-Barrera staining and immunostaining of beta-amyloid precursor protein (APP). In LFPI animals, the spontaneous alternation rate in the TMT remained below the random alternation rate (< 50%) on all post-injury test days. These animals also showed a transient tendency to choose only the right arm (ipsilateral to the injury) in the TMT at 6 hours and 24 hours after injury. Significant accumulation of APP was found widespread in the ipsilateral hemisphere including directly injured cortex, subcortical white matter, and hippocampal formation at 6 hours and 24 hours post-injury, while on day 7, the increased immunoreactivity of APP subsided. These results suggest that the widespread axonal degeneration of the white matter might contribute to the unilateral spatial neglect and memory deficit in the acute stage after LFPI.

Amyloid beta-Protein Precursor↗

PubMed2006

Long-term cognitive and neuropsychological symptoms after global cerebral ischemia in Mongolian gerbils.

The objective of this study was to establish a rodent model of vascular dementia that showed long-term cognitive and neuropsychological deficits, and to correlate those behavioral deficits with the patterns of ischemic lesions, thus providing a platform for future testing of potential therapeutic agents. In Mongolian gerbils, either 5-minute single bilateral common carotid artery occlusion (SBCCAO) or repetitive bilateral common carotid artery occlusion (two 7-minute occlusions, RBCCAO) was induced, and the behavioral deficits were evaluated using 2 tests: a modified open-field test with an escape zone to evaluate changes in anxiety and locomotor activity, and a T-maze test to assess cognitive dysfunction. SBCCAO did not induce anxiety changes but caused transient locomotor hyperactivity and mild cognitive deficits. Only pyramidal neuronal death was found in the bilateral CA1 sector of the hippocampus following SBCCAO. In contrast, RBCCAO induced persistent locomotor hyperactivity, reduced anxiety, and caused severe cognitive deficits at 4 weeks post-ischemia. RBCCAO caused significant atrophy associated with diffuse selective neuronal death in the bilateral cerebral cortex and caudate nucleus, as well as the CA1 region. The repetitive ischemia model appears to be a potentially useful platform for the long-term analysis of cognitive and neuropsychological symptoms associated with vascular dementia.

Animals↗

PubMed2006

The effect of renal ischemia-reperfusion injury on expression of RAE-1 and H60 in mice kidney.

INTRODUCTION: NKG2D, an activating receptor, may trigger NK and CD8+ T cells. Ligands for NKG2D and major histocompability complex class I chain-related antigen (MIC) have been reported to be expressed in rejected human renal allografts. MIC-NKG2D engagement may induce natural killer (NK) cell activation providing T-cell costimulation. We hypothesized that this interaction between innate and adaptive immunity may occur during kidney ischemia-reperfusion injury (IRI). METHODS: Male C57BL/6 mice after right renal resection were subjected to 35 minutes of left renal ischemia: the ischemic group. Sham-operated mice underwent the same protocol without vascular occlusion. The sham and ischemic kidneys were removed at 2 to 7, 10, 14, or 28 days postoperatively. The normal, sham, and ischemic kidney tissues were collected for reverse-transcriptase polymerase chain reaction, and immunohistochemistry analysis of MIC homologues in mice (Rae-1 and H60). RESULTS: Compared with no expression in sham control and normal kidneys, IRI caused mRNA expression of Rae-1 from 2 to 10 days postoperatively and protein expression of Rae-1 from 2 to 14 days postoperatively in ischemic kidneys. We observed no expression of H60 mRNA or protein in any kidney. CONCLUSION: H60 transcripts have been reported to be present in the BALB/c background but not in C57BL/6. We found IRI did not cause H60 mRNA on protein expression in C57BL/6 kidneys. Rae-1 was absent in normal C57BL/6 kidneys. The IRI-induced expression of the NKG2D ligand, Rae-1, might activate NK and CD8+ T cells. Our results suggested that MIC may be an important link between innate and adaptive immunity in kidney IRI.

Animals↗

PubMed2006

Hypoxia/reoxygenation up-regulated the expression of death receptor 5 and enhanced apoptosis in human hepatocyte line.

OBJECTIVES: Ischemia-reperfusion injury (IRI) is a key factor that contributes to early and late dysfunction of liver graft. Although we have known that hepatocytes express death receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), the effects of TRAIL on hypoxia/reoxygenation (H/R)-mediated apoptosis are unclear. This study sought to examine the effects of H/R on TRAIL cytotoxicity, as a cause of primary hepatic graft dysfunction, delayed graft refunction, and chronic graft dysfunction. METHODS: Using an hepatocyte H/R model in vitro to mimic IRI in the grafted liver, normal human hepatocytes HL-7702 were exposed to hypoxia for 5 hours then reoxygenated for 0, 2, 4, 6, or 20 hours. In another experiment, hepatocytes were exposed to hypoxia for 0, 2, 4, 8, or 20 hours. Expressions of TRAIL-R2/Death receptor 5 (DR5) mRNA were measured by semiquantitative reverse-transcriptase polymerase chain reactions. After 16 hours of hypoxia, human hepatocytes were treated with TRAIL in different concentrations for 5 hours. The death of hepatocytes was confirmed by flow cytometer and methyl thiazolyl tetrazolium analysis. RESULT: After 5-hour hypoxia, the expressions of DR5 mRNA increased at all times of reoxygenation. DR5 mRNA was up-regulated from 0 hour after reoxygenation, reaching a peak value at 2 hours after reoxygenation compared with the normoxia cultured cells. Moreover, DR5 mRNA was up-regulated gradually following prolonged hypoxia. TRAIL-mediated cell killing was concentration-dependent being greater in the hypoxia treatment group compared to the normoxia group. CONCLUSIONS: H/R up-regulated the expression of DR5 and enhanced TRAIL-mediated apoptosis in an human hepatocyte line. The TRAIL pathway might play a critical role in hepatocyte apoptosis induced by IRI.

Apoptosis↗

PubMed2006

Selective depletion of activated T cells by recombinant immunotoxin containing anti-CTLA-4 single-chain fragment of variable antibody and N-terminal fragment of perforin.

We constructed a novel immunotoxin, hS83P34, by fusing the fragment containing the N-terminal 34 amino acids of human perforin to the C-terminal of humanized anti-CTLA-4 single chain fragment of variable antibody. In vitro cytotoxicity assays demonstrated that CTLA-4-positive activated human T cells and 6T-CEM were sensitive to hS83P34, while CTLA-4-negative resting T cells and endothelial cell ECV-304 were resistant to hS83P34. The IC50s of hS83P34 for activated T cells and 6T-CEM were about 0.2 micromol/L and 1.0 micromol/L, there was no obvious cytotoxicity of ECV-304 as detected at 8 micromol/L of hS83P34. In tumor graft rejection models, after treatment with 1.2 mg/kg immunotoxin every day for 12 days, the transplanted tumor cells were rescued by immunotoxin. The tumor weights of grafts of the rejection control group, nonrejection control group, and test group were 0.006 +/- 0.014 g, 0.261 +/- 0.048 g, and 0.135 +/- 0.056 g, respectively. In the early 3 days posttransplantation, there were a lot of CD4- and CD8-positive T cells infiltrating into the tumor grafts of the rejection control group, while only a few T cells were detected in the tumor grafts of the test group. According to these results, we concluded that immunotoxin hS83P34 selectively depleted activated T cells in vitro as well as in vivo in an acute rejection model.

Antigens, CD↗

PubMed2006

Effect of cyclosporine on expression of MIC in human hepatocytes.

OBJECTIVES: The human major histocompatibility complex class I chain-related antigen A, B (MICA, B) are believed to be "cell stress sensors," which encode stress-inducible surface proteins that bind to NKG2D, an activating receptor of NK, alpha beta and gamma delta T cells. While cyclosporine (CsA) has improved patient and graft survival rates following solid organ transplantation, its clinical use is often limited by acute and chronic toxicity. Besides ischemia-reperfusion injury, which could up-regulate the expression of MIC, the toxicity of CsA may also be another violent stress factor. The purpose of this study was to investigate whether CsA can affect the expression of MIC. METHODS: Various doses of CsA (5 or 20 microg/mL) was added to cultured human hepatocytes for 1, 3, 5, 8, or 24 hours. The expression of MIC was measured by immunofluorescence using a laser scanning confocal microscope. RESULTS: CsA (5 or 20 microg/mL) upregulated MIC protein expression in hepatocytes at 1 hour, peaking at 3 hours, and persisting until 5 hours (P < .05), returning to normal levels after 8 to 24 hours (P > .05). CONCLUSION: This study reported that CsA up-regulated MIC expression on human hepatocytes. MIC, as a "cellular stress biomarker," may play a significant role in activating NK, T, and B lymphocyte responses associated with transplantation. This study suggested that immunosuppressants produce deterioration in organ function in transplantation.

Cells, Cultured↗

PubMed2006

Nuclear translocation of SMAD3 may enhance the TGF-beta/SMADS pathway in high glucose circumstances.

OBJECTIVES: Posttransplant diabetes mellitus is one of the most frequent complications after kidney transplantation. It is considered to be one cause of chronic allograft nephropathy. This study sought to investigate the effects of high glucose on the expression and nuclear translocation of Smad3, which is an important signal mediator involved in the fibrotic signal pathway. METHODS: The established rat renal mesangial cell line HBZY-1 was cultured in medium with various concentrations of glucose (4.5 mg/mL, 9.0 mg/mL, or 13.5 mg/mL), which was collected at 7, 14, or 21 days. The total expression of Smad3, including both inner and outer nucleus proteins was examined by Western blot analysis. The nuclear translocated Smad3, representing only the inner nucleus protein, was detected by immunofluorescence staining observed under a laser confocal scanning microscope. RESULTS: No significant difference in the total Smad3 expression was demonstrated by Western blot analysis among the three groups of HBZY-1 cells at various concentration of glucose after 7, 14, or 21 days. There was no fluorescence detected in the nucleus at day 7 by immunofluorescence staining; however, robust positive expression of Smad3 was detected at days 14 and 21. CONCLUSION: As a restricted Smads member, Smad3 protein might not be upregulated in the presence of high glucose. However, with prolonged culture time, Smad3 translocates from cytoplasm to nucleus, which may be a pivotal step in the fibrotic signal pathway.

Animals↗

PubMed2006

The C-terminal peptide of connective tissue growth factor blocks the full molecule binding to tubular epithelial cell.

OBJECTIVES: Interstitial fibrosis is a critical pathologic change in chronic allograft nephropathy. The cytokine connective tissue growth factor (CTGF, also CCN2) plays an important role in epithelial-mesenchymal transformation (EMT) of tubular epithelial cells to renal interstitial fibrosis. The hexadeca-peptide within the C-terminal of CTGF (named P2) contains the unique binding domain of CTGF to its potential receptor, integrin alphavbeta3. This study examined whether P2 bound preferentially to the receptor and served as an inhibitor of CTGF. METHODS: All studies used an established rat kidney tubular epithelial cell line NRK-52E. Chemically synthesized P2 was purified, and some of it labeled with FITC. The affinity of CTGF or P2 to NRK-52E cells was examined by a solid-phase cell adhesion assay. Competitive binding between P2 and CTGF to NRK-52E cells was examined with flow cytometric analysis. RESULTS: Both P2 and CTGF bound to the NRK-52E cells, mediating cell adhesion. When the cells were incubated in the mixture of P2 and CTGF, P2 bound to the cells preferentially. Furthermore, when cells were preincubated with excessive CTGF, it became difficult for subsequent P2 binding to occur. CONCLUSIONS: P2 and CTGF seemed to bind to cell membranes at the same binding domain. P2 competitively blocked CTGF binding, acting as a CTGF inhibitor.

Animals↗

PubMed2006

Construction and characterization of a cDNA library from liver tissue of Chinese Banna minipig inbred line.

A xenograft that performs efficient functions is an essential premise for successful xenotransplantation. Our early study indicated that Chinese Banna minipig inbred line (BMI) was an ideal xenograft donor. However, the activities of some proteins synthesized by the BMI liver are different from the human, which could lead to functional disorders in coagulation, fibrinolysis, and anticoagulation after liver xenotransplantation. Therefore, it is important to investigate the genetic background of protein incompatibility and to provide new strategies for gene manipulation. In this study we constructed a cDNA expression library using BMI liver tissue to obtain an understanding of nucleic acid and protein differences between the two species. We extracted total RNA and purified mRNA of the liver tissue from one of the sixteenth inbred generation of BMI/JS 151 substrain. After double-strand cDNA synthesis, we fractionated it on a CHROMA APIN-400 column; ligated the longer than 500bp cDNA into a ZAP Express Vector; and performed a lambda: phage packaging reaction, library amplification, and titer. We randomly picked 12 plaques and tested the length of inserts. The titers of the primary and amplified libraries were 1.0 x 10(6) pfu/mL and 5.0 x 10(9) pfu/mL, respectively. The percentages of recombinants were 97.0% in the primary library and 98.0% in the amplified library. The lengths of most inserts were between 750 bp and 2.0 kb. Thus, we successfully constructed a cDNA expression library from BMI liver tissue. Using the library, we hope to get a full-length cDNA of some important genes and conduct further studies on porcine liver function in xenotransplantation.

Animals↗

PubMed2006

Expression of H60 on mice heart graft and influence of cyclosporine.

OBJECTIVES: It has been reported that the MHC class I chain-related antigen (MIC), a ligand of NKG2D, an activating receptor of natural killer cells, is expressed on rejected renal allografts. This study investigated whether heart transplantation induced expression of a homologue of MIC (H60) in outbred Kun Ming (KM) mice, widely used in China, and whether CsA had an influence on the process. METHODS: Forty-eight KM female mice were divided into untreated and CsA-treated groups, after cervical heart allotransplantation. Grafts were harvested 1, 3, 5, and 7 days postoperatively. H60 mRNA was analyzed by RT-PCR, and the protein detected by immunohistochemistry. RESULTS: Compared with no mRNA expression in the normal heart, H60 mRNA was observed at day 5 and upregulated on day 7 in the untreated group. It was detected on day 3, peaked on day 5, but was lower than untreated group, and decreased on day 7 in the CsA-treated group. H60 protein was detected in cardiocytes only on day 7 in the untreated group. CONCLUSION: Our study suggested that expression of the NKG2D ligand, H60, may activate natural killer cells playing a significant role in innate immunity associated with transplantation. The early expression of H60 mRNA on day 3 in the CsA-treated group might relate to the toxicity of CsA. The expression peaked on day 5 and decreased on day 7, possibly induced by CsA. The results suggested that H60 might be a new target for prevention of rejection.

Animals↗

PubMed2006

Yisheng injection decreases the expression of H60 and RAE-1 genes in ischemic mice liver.

OBJECTIVE: Major histocompatability complex class I chain-related antigen A, B (MICA, B) functions as ligands for human NKG2D receptors, which may play a role in graft rejection and cellular stress. In this study we explored the effect of ischemia/reperfusion injury (IRI) on the expression of H60 and RAE-1 (MICA, B homologues) in mice to study the protective effect of Yisheng injection (YS), an herbal preparation developed from traditional Chinese medicine. METHODS: Male BALB/c mice were divided into sham, ischemic, and YS-treated groups using 90 minutes of left liver lobe ischemia. Sham control mice underwent the same operation, but without vascular occlusion. In the treated group, YS (20 mg/kg) was given before ischemia and after reperfusion for 7 days. Liver samples collected at 7 days postoperation were used for real-time quantitative polymerase chain reaction analysis, Western blotting, and immunohistochemical assays. RESULTS: Compared with the sham group, H60 and RAE-1 mRNA levels were increased by sevenfold and 4.5-fold in the ischemic group, respectively. After YS treatment, they were reduced by 76% and 70%, respectively. Western blotting and immunohistochemical assays showed that there was absent or faint H60 and RAE-1 expression in sham liver, but they were apparently increased in ischemic liver; however, the expressions were significantly decreased in the presence of YS. CONCLUSIONS: Hepatic IRI significantly increased H60 and RAE-1 expression in mouse liver. YS treatment effectively reduced this increase, seeming to attenuate NKG2D-ligand-mediated immune responses caused by IRI. This may suggest a new concept to prevent IRI and graft rejection.

Animals↗

PubMed2006

Distribution of the alpha-gal epitope on adult porcine bone tissue.

INTRODUCTION: Bone xenografts from pig to human appear to be an alternative to resolve the shortage of bone autologous and allografts. However, the major obstacle of pig-to-human xenotransplantation is the interaction between human natural anti-Gal antibody and the alpha-Gal epitope abundantly expressed on pig endothelium. It was important to investigate the expression of alpha-Gal epitopes in porcine bone tissue to look for an ideal method to remove the alpha-Gal epitopes. METHODS: The cortical and trabecular bone were retrieved from five pigs. After the soft tissues and periosteum were removed, the blood and marrow cavity were cleaned with phosphate-buffered solution. All 5 mm x 5 mm x 5 mm samples were imbedded in paraffin and methyl methacrylate resin for histological sections. The mouse IgM M86 monoclonal antibody, which was highly specific for alpha-Gal epitopes, was used to document alpha-Gal epitope expression by immunostaining of tissues and immunofluorescence. RESULTS: Gal-positive immunostaining and immunofluorescence were observed on the surface of osteocytes and Haversian canals. There was a significant difference in Gal expression between cortical and trabecular bone tissues. There was no Gal expression in the extracellular matrix of bone. CONCLUSIONS: Major alpha-Gal epitopes were on the surface of osteocytes of porcine bone tissues. A method should be used to damage the osteocytes and eliminate the alpha-Gal epitopes to avoid the xenogenic rejection in xenotransplantation of porcine bone tissues.

Animals↗

PubMed2006

Intraperitoneal injection of oxygenated perfluorochemical improves the outcome of intraportal islet transplantation in a rat model.

A 50% to 75% early graft loss upon engraftment has been suggested to in intraportal islet transplantation (IPIT). Hypoxia in the portal vein contributes to graft loss in immediately posttransplantation. Herein we examined the effect on the outcome of IPIT of intraperitoneal oxygenated perfluorochemical (PFC) as an oxygen carrier. Isolated Lewis rat islets were transplanted into the portal vein of a chemically induced diabetic syngeneic rat. First, 1500 IEQ was determined to be the optimal dose in this study. When oxygenated PFC (group 1) was intraperitoneally injected following IPIT of 1500 IEQ, the success rate of transplantation was 5/6, in contrast to 1/6 when PFC with no oxygen was injected (group 2) and 1/6 in IPIT without PFC, respectively. The area under the glucose profile curve on intraperitoneal glucose tolerance tests on posttransplant day 28 in group 1 was significantly smaller than that for group 2. In conclusion, intraperitoneal oxygenated PFC improved the outcome of IPIT.

Animals↗

PubMed2006

A novel triple purge strategy for eliminating chronic myelogenous leukemia (CML) cells from autografts.

Imatinib-refractory chronic myelogenous leukemia (CML) patients can experience long-term disease-free survival with myeloablative therapy and allogeneic hematopoietic cell transplantation; however, associated complications carry a significant risk of mortality. Transplantation of autologous hematopoietic cells has a reduced risk of complications, but residual tumor cells in the autograft may contribute to relapse. Development of methods for purging tumor cells that do not compromise the engraftment potential of the normal hematopoietic cells in the autograft has been a long-standing goal. Since primitive CML cells differentiate more rapidly in vitro than their normal counterparts and are also preferentially killed by mafosfamide and imatinib, we examined the purging effectiveness on CD34(+) CML cells using a strategy that combines a brief exposure to imatinib (0.5-1.0 microM for 72 h) and then mafosfamide (30-90 microg/ml for 30 min) followed by 2 weeks in culture with cytokines (100 ng/ml each of stem cell factor, granulocyte colony-stimulating factor and thrombopoietin). Treatment with 1.0 microM imatinib, 60 microg/ml mafosfamide and 14 days of culture with cytokines eliminated BCR-ABL(+) cells from chronic phase CML patient aphereses, while preserving normal progenitors. This novel purging strategy may offer a new approach to improving the effectiveness of autologous transplantation in imatinib-refractory CML patients.

Antigens, CD↗