Establishment of an in vivo model for pediatric Ewing tumors by transplantation into NOD/scid mice.

Ewing tumors are a clinically heterogeneous group of childhood sarcomas that represent a paradigm for understanding solid tumor biology, as they are the first group of sarcomas for which a chromosome translocation has been characterized at the molecular level. However, the biologic organization of the tumor, especially the processes that govern proliferation, differentiation, and metastasis of primitive tumor stem cells is poorly understood. Therefore, to develop a biologically relevant in vivo model, five different Ewing tumor cell lines and primary tumor cells from three patients were transplanted into immune-deficient mice via intravenous injection. NOD/scid mice that carry a complex immune deficiency and thus nearly completely lack the ability to reject human cells were used as recipients. Overall, 26 of 52 mice (50%) transplanted with VH-64, WE-68, CADO-ES1, TC-71, and RM-82 cells and 4 of 10 mice (40%) transplanted with primary tumor cells engrafted. Moreover, primary cells that did not grow in vitro proliferated in mice. The pattern of metastasis was similar to that in patients with frequent metastases in lungs (62%), bone marrow (30%), and bone (23%). Using limiting dilution experiments, the frequency of the engraftment unit was estimated at 1 Ewing tumor-initiating cell in 3 x 10(5) VH-64 cells. These data demonstrate that we have been able to establish an in vivo model that recapitulates many aspects of growth and progression of human Ewing tumors. For the first time, this model provides the opportunity to identify and characterize primitive in vivo clonogenic solid tumor stem cells. This model will, therefore, be instrumental in studying many aspects of tumor cell biology, including organ-selective metastasis and tumor angiogenesis.

Expression of AC133 and CD117 on candidate normal stem cell populations in childhood B-cell precursor acute lymphoblastic leukaemia.

To identify residual candidate normal progenitor/stem cell populations in childhood B-cell precursor acute lymphoblastic leukaemia (ALL), expression of AC133 and CD117 was analysed on the leukaemic cell clone and on immature B-lineage-negative CD34+CD19- bone marrow cells. 10/25 patients (40%) had no detectable expression of AC133 within the leukaemic cell clone. 24/26 patients (92%) lacked expression of CD117 on the leukaemic blast cell population. In contrast, a distinct AC133-positive cell population was found in 8/8 children with AC133-negative ALL and a CD117-positive cell population could be identified in 12/12 children with CD117-negative ALL, within the CD34+CD19- progenitor/stem cell compartment. These observations provide further evidence that in B-cell precursor ALL, unlike in acute myelogenous leukaemia, it may be possible to distinguish residual normal progenitor/stem cells from the leukaemic cell clone.

Flow cytometric identification of candidate normal stem cell populations in CD45-negative B-cell precursor acute lymphoblastic leukaemia.

CD45-negative B-cell precursor acute lymphoblastic leukaemia (ALL) provides a unique model to study the stem cell compartment in ALL as leukaemic CD34-positive cells, unlike their normal counterparts, do not express CD45. By increasing the number of events analysed to 10(6), storing only the events in the region of interest (storage gate), using appropriate isotype controls and stringent washing procedures, a flow cytometric protocol was established to characterize rare CD34+ CD19- events. In eight of 12 patients (67%) with CD45-negative B-cell precursor ALL, a distinct CD34+ CD19- CD45+ candidate normal stem cell population could be detected. In one patient analysed by four-colour staining, the CD34+ CD19- CD45+ cells, unlike the CD45-negative leukaemic cells, expressed CD117 (c-kit), providing further evidence that these cells represent residual nonleukaemic normal cells. By multiparameter analysis, this population of candidate normal stem cells could be separated from contaminating leukaemic CD34+ CD19- CD45- cells, which were detected in 11 of the 12 patients within the CD34+ CD19- compartment.

Good engraftment of B-cell precursor ALL in NOD-SCID mice.

BACKGROUND: In vivo models for human B cell precursor ALL have been established by transplanting human leukemic cells onto immune-deficient SCID mice. High risk and relapsed leukemias engraft very well in these mice, however, good prognosis pediatric ALL often grow poorly if at all. Recently a new, even more immune-deficient mouse strain has been bred by crossing the scid mutation onto the NOD mouse background. As these NOD-SCID mice have been shown to be better recipients for human myeloid cells the goal of this study was to test these mice as hosts for human acute lymphoblastic leukemia cells. PATIENTS AND METHODS: Bone marrow or peripheral blood cells from/pediatric and one adult patient with B-cell precursor ALL were transplanted onto immune-deficient NOD-SCID mice according to established protocols. MAIN RESULTS: ALL cells from 6 out of the 8 patients (75%) successfully engrafted the NOD-SCID mice and from 4 patients (50%) led to an extensive leukemic infiltration in the murine marrow (> 10%). High level human cell engraftment could be demonstrated by flow cytometry, Southern blot analysis and cytology. By cytology and immunophenotype the leukemia in the mice was indistinguishable from the original leukemia in the patients. The presence of few human eosinophils in the marrow of highly engrafted mice indicates minimal coengraftment of residual normal cells. Development of overt leukemia in the mice after transplantation of cells from different patients varied between 1.5 and 7 months. Interestingly and in contrast to myeloid cells, conditioning of the mice by sublethal irradiation was not necessary for successful engraftment. Limiting dilution experiments with leukemic blasts from one patient showed that as few as 10000 cells were sufficient to transfer the leukemia onto NOD-SCID mice. CONCLUSIONS: NOD-SCID mice are sensitive recipients for human ALL xenografts.

A new bioassay to study contractile and relaxant effects of PGE2 on perfused guinea pig trachea.

The aim of this study was to investigate the effects of PGE2 under different experimental conditions. By a newly developed imaging bioassay system, changes of the diameter of isolated perfused guinea-pig tracheal tubes were continuously measured. The results were compared with those of a conventional ring bioassay system. The effect of PGE2 was examined in epithelium-intact and-denuded tubes. The potency of PGE2 applied to the inner surface of tube preparations was significantly enhanced by about six-fold in epithelium-denuded compared with epithelium-intact tubes. In rings there was no difference. The results suggest a potential role of epithelium in assessment of airway reactivity in vivo. The study demonstrates, that the effects of PGE2 depend critically on the experimental approach. PGE2 (0.01 nmol 1(-1)-1 mumol 1(-1)) contracted tracheal tubes, but resulted in relaxation of ring preparations under basal tone. The spontaneous tone of guinea pig trachea can be reduced with the cyclooxygenase inhibitor diclofenac. Pretreatment of the rings with diclofenac reversed the relaxation into contraction. Preincubation of tube preparations with diclofenac had no effect on basal tone, indicating that there was no inherent spontaneous prostanoid-dependent tone or the mediators may be washed out at a relatively fast rate and, therefore, tone may not be apparent.

[Flow cytometric characterization of maturation processes and primitive stem cell population in pre-B-cell ALL in childhood]. / Durchflusszytometrische Charakterisierung von Reifungsvorgängen und stammzellnahen Zellpopulationen bei der B-Vorläuferzell-ALL im Kindesalter.

Bone marrow and peripheral blood from children with acute lymphoblastic leukemia was analyzed by flow cytometry to assess leukemic cell differentiation and to characterize the profile of cell surface marker expression on rare CD34+ cell populations. The goal of this study was to determine if patterns of cell surface antigens could be identified on CD34+ subpopulations which may allow distinction between normal and leukemic stem cells. Expression of the progenitor cell antigen CD34 on leukemic blasts was very heterogeneous and varied between 0.5 and 100% in 20 patients analyzed in this study. In cALL and pre-B-ALL, a variable percentage of the leukemic cells coexpressed CD20 in addition to CD10. Only in one case, differentiation characteristic for normal B cell development with coordinated downregulation of CD10 with increasing expression of CD20 was observed. By analysing 5 x 10(6)-1 x 10(6) cells, a CD34+ cell population could be identified in 8 out of 8 patients which did not express CD19 and comprised less than 0.1% of all bone marrow or peripheral blood cells. Within this population, there was differentiation from primitive CD34-CD38- to more mature CD34+CD38+ cells. In 4 of these patients, an additional CD34+ population with low expression of CD19 (CD34+CD19lo) was detected. The lack of CD45 expression on the leukemic cells of 2 patients was used as a marker for the leukemic cell clone. In both patients, the CD34+CD19- cells did express CD45 while CD34+CD19lo/+ cells were CD45 negative. This suggests that the CD34+CD19lo cells were part of the leukemic clone and that the CD34+CD38-CD19- cells may represent residual normal primitive hematopoietic cells. In conclusion, flow cytometry allowed identification of primitive CD34+ cell populations in children with ALL, which can now be functionally characterized by transplantation onto immune-deficient mice.

Differential effects of two calcium channel blockers on bronchial smooth muscle.

The effects of the new Ca(2+)-entry blocker SK&F 96365 (1-¿beta-[4-methoxyphenyl)propoxy]-4-methoxy-phenethyl¿-1H- imidazole hydrochloride, [sequence: see text] CAS 130495-35-1) and nifedipine on the contractile response of guinea-pig trachea to KCl and histamine are described. Diameter changes of cannulated, perfused tubes of guinea-pig trachea were recorded by a computerized video-microscopy bioassay system. In order to determine whether the capacitive calcium entry is the major mechanism in the guinea-pig trachea, the effects of SK&F 96365, an antagonist of the second-messenger operated channel, were studied. The results of experiments in Ca(2+)-free medium containing EGTA (0.1 mmol l-1) indicate that KCl- and histamine evoked contractions are mainly due to an influx of extracellular Ca2+ rather than due to release from intracellular stores. SK&F 96365 (10 mumol l-1) showed a more effective antispasmogenic activity on histamine-induced contraction of guinea-pig trachea than the voltage-operated channel (VOC)-blocker nifedipine (10 mumol l-1). Nifedipine is only effective in inhibiting KCl induced contraction. These results may have significance for the use of agents such as SK&F 96365 in the treatment of asthma.