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N G Testa

Publications and source records attributed to N G Testa.

At least 19 recordsLinked to original sources
PubMed1998

Interaction of prostate epithelial cells from benign and malignant tumor tissue with bone-marrow stroma.

BACKGROUND: Metastases of prostate cancer form selectively within the skeleton. To understand this metastatic spread, we studied the ability of prostate epithelial cells to grow and proliferate within the bone marrow, using primary coculture. METHODS: Prostate epithelia and fibroblasts were prepared from men with benign prostatic hyperplasia (n = 13) and cancer of the prostate (n = 10). Confluent cultures of bone-marrow stroma or fibroblast controls were prepared in 96-well plates, and identical plates were treated with detergent to expose the extracellular matrix of the cells. Epithelial cells were seeded onto either cells or matrix, and their growth characteristics were determined by counting increases in colony size and number over time. Further experiments evaluated the effects on epithelial growth when cells were exposed to media conditioned by these stroma, using an MTT assay. RESULTS: Results showed that for epithelial cells derived from malignant (or benign) tissue, the median value of the total area of colonies formed on bone-marrow stroma was 2.1 (benign, 2.6) mm2, in contrast to 0.3 (benign, 0.4) mm2 or 0.25 (benign, 0) mm2 when these cells were cocultured with fibroblasts from benign or malignant prostates, respectively. Statistics indicated that growth was significantly greater on bone-marrow stroma than on control stroma (P < 0.005). However, no significant stimulation of epithelial cell growth was seen when these epithelial cells were cultured on extracellular matrix from bone-marrow stroma or when exposed to bone-marrow stroma-conditioned media in comparison to fibroblast controls. No statistical differences were found between the formation of colonies from malignant tissue in comparison to benign. CONCLUSIONS: This system allows the investigation of bone-marrow stroma colonization by primary prostate epithelial cells, and could be developed for the study of metastasis.

Bone Marrow Cells↗
PubMed1998

Accelerated telomere shortening in young recipients of allogeneic bone-marrow transplants.

BACKGROUND: The establishment of donor-derived haemopoiesis in the recipients of allogeneic bone-marrow transplants (BMT) involves extensive proliferation of haemopoietic stem cells. The biological consequences of this replicative stress are ill defined, but any "ageing" effect would carry the risk of an increased frequency of clonal disorders during later life. We compared blood-cell mean telomere lengths in donor/recipient pairs. METHODS: Mean telomere length was calculated by in-gel hybridisation to leucocyte DNA from 56 normal individuals aged 0-96 years, and from 14 consecutive BMT recipients (aged 2-14 years) plus their respective donors (aged 2-46 years). Engraftment was confirmed by variable numbers of tandem repeats (VNTR) or gender analysis. FINDINGS: On average, blood-cell telomeres of transplant recipients were 0.4 kb (95% CI -0.2 to -0.6) shorter than those of their respective donors. This degree of telomere loss is equivalent to a median of 15 years' (range 0-40) ageing in the healthy controls. INTERPRETATION: The kinetics of haemopoietic engraftment impose replicative stress on the haemopoietic stem cells, resulting in a pronounced ageing effect, which may be sufficient to accelerate the onset of clonal haemopoietic disorders usually associated with later life. Monitoring of haemopoietic status in BMT recipients as time since BMT increases will be important. Assessment of transplant protocols under development in terms of their effects on telomere shortening is also indicated.

Adolescent↗
PubMed1998

Factors which affect the CFU-GM content of the peripheral blood haemopoietic progenitor cell harvests in patients with acute myeloid leukaemia.

Autologous peripheral blood haemopoietic stem cells (PBSC) were harvested from 30 patients with de novo acute leukaemia, 29 of whom had entered remission following standard chemotherapy. Correlation of CD34+ cells/kg to CFU-GM/kg in the harvests was good (correlation coefficient = 0.72, P < 0.001). We demonstrated significant associations between the CFU-GM content of the harvest and the following: time to platelets >50 x 10(9)/l post final induction course (P < 0.001), days to harvest from day 1 of intensification/mobilization (correlation coefficient = -0.73, P < 0.001), platelets >20 x 10(9)/l at time of harvest (P = 0.02), time to WBC >1.0 x 10(9)/l post intensification/mobilization (correlation coefficient = -0.70, P < 0.001), and WBC on day of harvest (correlation coefficient = 0.60, P < 0.001). In contrast, we found no relationship between the CFU-GM content of the harvest and patient age up to 65 years, presence of absence of coexistent features of trilineage myelodysplasia at diagnosis, number of induction courses to remission or total number of courses of chemotherapy prior to intensification/mobilization. Haemopoietic recovery after reinfusion of PBSC was highly correlated to the number of CFU-GM infused (neutrophils >0.5 x 10(9)/l rs = -0.72, P = 0.001; platelets >20 x 10(9)/l unsupported rs = -0.71, P = 0.001). Our results show that the number of induction courses received, and thus exposure to cytotoxic agents received, made no significant difference to subsequent CFU-GM harvest content. We collected superior harvests from those patients with faster platelet recovery following mobilization therapy. We also found that faster platelet recovery following the final induction therapy was a better predictor of the CFU-GM harvest following mobilization than was the neutrophil recovery following final induction.

Acute Disease↗
PubMed1997

Investigation of mammary epithelial cell-bone marrow stroma interactions using primary human cell culture as a model of metastasis.

A model has been established using primary human cell culture to study the cell biology of breast cancer metastasis to bone marrow. Mammary epithelia were obtained in single cell suspension from tumour (macroscopically involved), benign (macroscopically uninvolved) and normal (reduction mammoplasty) breast tissue as well as from locally involved lymph nodes. Stromal layers were generated from long-term cultures of human bone marrow or from mammary fibroblasts derived from normal or malignant tissue. The interaction between epithelia and stroma has been studied in terms of adhesion of the epithelia to the stroma and their subsequent growth in co-culture. Our results show that when assayed up to 9 hr after plating, epithelial cells from malignant tissue (14 primary tumours and 9 metastases in lymph nodes) displayed a significant preference for adhesion to bone marrow stroma compared with mammary fibroblasts. In contrast, epithelial cells from 4 normal and 2 of 4 benign samples showed no significant preferential adherence. Subsequent co-culture of mammary epithelia with each of the 3 stromal layers revealed that under serum-free, in vitro conditions, bone marrow stromal layers did not provide an advantageous environment for colony growth, in contrast to their ability to provide a preferential substratum for adhesion.

Animals↗
PubMed1997

Primary prostatic epithelial cell binding to human bone marrow stroma and the role of alpha2beta1 integrin.

Prostate cancer selectively metastasises to the bone. To investigate the importance of prostate epithelial cell adhesion to bone marrow cells in this process we examined the binding of human primary prostatic epithelial cells (PEC) to human bone marrow stromal cultures (BMS). We found that PEC derived from both malignant and benign tissue showed greater adhesion to BMS than to benign prostatic fibroblasts (median difference was 340% and 200% respectively), skin fibroblasts or plastic tissue culture plates. Adhesion to BMS grown from the bone marrow of patients with prostatic skeletal metastases was no different from those grown from normal bone marrow. The role of integrin molecules in these cell interactions was determined. Collagen type I and fibronectin were found to increase PEC adhesion whereas vitronectin and laminin did not. Inhibition studies demonstrated that although there was heterogeneity between samples, antibodies against the integrins alpha2 and beta1 consistently inhibited PEC binding to BMS. This result was more marked for PEC derived from malignant tissue. However studies investigating the effects of disintegrins and anti-alpha3 and anti-alpha5 integrins indicated that for a percentage of patients these integrins and RGD (arginine, glycine, aspartamine)-dependent binding pathways were also involved. In summary, the results indicate that BMS are adherent to primary PEC derived from both malignant and benign tissue. The integrin alpha2beta1 is a major contributor to this interaction.

Animals↗
PubMed1997

Autografting in Philadelphia (Ph)+ chronic myeloid leukaemia using cultured marrow: an update of a pilot study.

Incubation of CML marrow in long-term culture (LTC) conditions may result in selection of normal (Ph-) LTC-initiating cells (LTC-IC) as early as 10 days, and in production of Ph- clonogenic cells and mature end cells within 5 weeks. This was the rationale for using marrow cells from 10-day-old LTC to autograft nine chronic phase CML patients, ineligible for HLA-matched sibling donor transplant, and who were selected on the basis of a pre-transplant screening LTC test. Of the transplanted patients three died; two of graft failure and one of therapy-related toxicity with 97% Ph- cells 16 months following the autograft. The reconstituting haemopoietic cells in the seven engrafted patients were 100% Ph- in four, > or = 90% Ph- in two and 71% Ph- in the seventh, with a duration of complete cytogenetic response of 6-12 months. Three patients reverted to chronic phase and 100% Ph+ haemopoiesis 27-36 months post-autograft. The other three patients remain in continuous haematological remission with 22% Ph- cells in one and complete cytogenetic remission in the other two 3-4 years post-autograft. IFN therapy was generally introduced on the first evidence of recurrence of Ph+ cells or of cytogenetic deterioration. Further strategies to modulate immune surveillance in vivo may improve the outcome of cultured marrow autografts which give an initial and rather prolonged bias towards Ph- haemopoiesis.

Adult↗
PubMed1997

High-dose granulocyte-colony stimulating factor (G-CSF) in vitro induces the growth of high proliferative potential colony forming cells (HPP-CFC) in patients undergoing blood stem cell mobilization.

We evaluated the role of high-dose granulocyte colony stimulating factor (G-CSF) in vitro, in inducing the generation of high-proliferative potential colony forming cells (HPP-CFC), from either mononuclear cells or purified CD34+ cells. Both normal controls and patients undergoing peripheral blood stem cell (PBSC) mobilization and transplantation were studied. In serum-driven agar cultures, G-CSF stimulated the proliferation of HPP-CFC in a dose dependent manner (r = 0.92). The number of HPP-CFC was four-fold greater in mobilized patients than in normal controls. Purified CD34+ cells yielded 11-fold more colonies than mononuclear cells. HPP-CFC from mobilized patients showed replating capacity, giving rise to secondary colonies of more mature appearance. In serum-free cultures, the effect of G-CSF appeared to be mediated by synergistic interaction with stem cell factor. Our results suggest that G-CSF stimulates primitive hematopoietic cells that are detectable in increased amounts in patients receiving mobilization therapy. Therefore, determination of G-CSF induced HPP-CFC could be a useful tool in the evaluation of mobilization strategies.

Antigens, CD34↗
PubMed1997

Measurement of long-term culture initiating cells (LTC-ICs) using limiting dilution: comparison of endpoints and stromal support.

Although much progress has been made in defining primitive cell phenotypes using flow cytometry and clonogenic assays, the direct study of marrow repopulating cells remains elusive. Long-term culture initiating cells (LTC-ICs) are arguably the most primitive human hematopoietic cells detectable by in vitro functional assays. Two endpoints have been reported for scoring LTC-ICs in limiting dilution assays. The first endpoint described was the generation of colony forming cells (CFCs) after 5 to 8 weeks of culture. An alternative method for scoring the LTC-IC assay is to identify cobblestone area forming cells. In the present study, estimations of LTC-IC frequency were made by measuring both endpoints and by comparing LTC-IC frequencies measured using limiting dilution assays of normal human bone marrow stroma with the measurements for murine bone marrow stromal cell line M2-10B4. For assays established on normal human bone marrow stroma, there was an equivalence between the two endpoint measures. Likewise, there was an equivalence between the two types of stroma when scoring CFC generation after 5 weeks. However, a consistently higher frequency of LTC-ICs was estimated when scoring cobblestone areas compared with that found when scoring CFC generation on the M2-10B4 stroma (p < 0.0001). Although the murine bone marrow stromal cell line M2-10B4 remains a very useful and consistently reliable alternative to normal human bone marrow stroma, these data indicate that the LTC-IC populations defined by scoring cobblestone areas or by measuring the generation of CFCs on this cell line are, in contrast to those measured using bone marrow stroma, not identical.

Animals↗
PubMed1996

Increased numbers of long-term culture-initiating cells in the apheresis product of patients randomized to receive increasing doses of stem cell factor administered in combination with chemotherapy and a standard dose of granulocyte colony-stimulating factor.

Long-term culture-initiating cells (LTC-IC) are arguably the most primitive human hematopoietic cells detectable by in vitro functional assays. We have investigated the mobilization of these cells into the blood of patients with ovarian carcinoma randomized to receive granulocyte colony-stimulating factor (G-CSF; 5 micrograms/kg) plus different doses of stem cell factor (SCF; c-kit ligand) after chemotherapy or G-CSF alone after chemotherapy. We have shown a significant SCF dose response for the mobilization of LTC-IC, with a 5.8-fold increase in LTC-IC mobilization in those patients receiving chemotherapy, G-CSF, and 20 micrograms/kg of SCF, the highest dose used, compared with the patients receiving chemotherapy and G-CSF alone. We have shown a threefold increase in CD34+ cells and up to a 64-fold increase in CD34+/33- cells was seen in patients treated with chemotherapy, G-CSF, and 20 micrograms/kg of SCF compared with those patients treated with chemotherapy and G-CSF alone. However, significant numbers of CD34+/38- cells were only found in the patients receiving 20 micrograms/kg of SCF as part of their mobilization regimen. Patients receiving chemotherapy plus G-CSF and SCF have enhanced mobilization of primitive cells and of the more committed progenitor cells compared with those patients receiving chemotherapy followed by G-CSF alone.

Adolescent↗
PubMed1996

Extensive amplification of single cells from CD34+ subpopulations in umbilical cord blood and identification of long-term culture-initiating cells present in two subsets.

CD34+ cord blood cells were isolated with immunomagnetic beads and fractionated by fluorescence-activated cell sorting (FACS) into three subpopulations: CD34+38+DR+, CD34+38-DR+ and CD34+38-DR-, using antibodies specific for these cell surface markers. Cells from each of the three subsets were plated as single cells in serum-free medium supplemented with a combination of growth factor and individual cells were monitored for proliferation and the capacity to form colony-forming cells. Single cells from the CD34+38+DR+ subset showed the lowest expansion capacity, generating up to 1.1 x 10(6) cells at five weeks, while individual cells from both the CD34+38-DR+ and CD34+38-DR- subsets could be expanded up to 1.8 x 10(6) and 9.2 x 10(6) cells, respectively, over a period of six weeks. The different subpopulations also generated colony-forming cells which gave rise to erythroid, myeloid and erythroid/myeloid colonies. CD34+38-DR+ cells generated large numbers of colonies within two weeks in liquid culture, but this rapidly declined. Generation of lineage-committed colony-forming cells was better sustained in the CD34+38-DR- population and continued for up to six weeks in culture. Overall, the generation of colony-forming cells declined with time in culture, although the cell numbers continued to expand. However, when the same populations were plated on irradiated bone marrow stroma, both the CD34+38-DR+ and the CD34+38-DR- cells were capable of producing granulocytemacrophage colony-forming cells (GM-CFCs) for 10 to 12 weeks. As hemopoiesis was sustained for almost three months, it appears that these populations were significantly enriched in long-term culture-initiating cells (LTC-ICs). Although both populations generated GM-CFCs, the CD34+38-DR- cells sustained production of higher numbers of colony-forming cells than the CD34+38-DR+ population. These results demonstrate that cells from cord blood can be efficiently monitored at the single-cell level for proliferation, expansion and colony-forming capacity. Furthermore, at least two populations of LTC-ICs can be distinguished in cord blood CD34+38- cells by the differential expression of the HLA-DR antigen.

ADP-ribosyl Cyclase↗
PubMed1996

A study of ovarian cancer patients treated with dose-intensive chemotherapy supported with peripheral blood progenitor cells mobilised by filgrastim and cyclophosphamide.

We have shown that large numbers of haemopoietic progenitor cells are mobilised into the blood after filgrastim [granulocyte colony-stimulating factor (G-CSF)] alone and filgrastim following cyclophosphamide chemotherapy in previously untreated patients with ovarian cancer. These cells may be used to provide safe and effective haemopoietic rescue following dose-intensive chemotherapy. Using filgrastim alone (10 micrograms kg-1), the apheresis harvest contained a median CFU-GM count of 45 x 10(4) kg-1 and 2 x 10(6) kg-1 CD34+ cells. Treatment with filgrastim (5 micrograms kg-1) following cyclophosphamide (3 g m-2) resulted in a harvest containing 66 x 10(4) kg-1 CFU-GM and 2.4 x 10(6) kg-1 CD34+ cells. There was no statistically significant difference between these two mobilising regimens. We have also demonstrated that dose-intensive carboplatin and cyclophosphamide chemotherapy can be delivered safely to patients with ovarian cancer when supported by peripheral blood progenitor cells and filgrastim. Carboplatin (AUC 7.5) and cyclophosphamide (900 mg m-2) given at 3 weekly intervals with progenitor cell and growth factor support was well tolerated in terms of haematological and systemic side-effects. Double the dose intensity of chemotherapy was delivered compared with our standard dose regimen when the treatment was given at 3 weekly intervals. Median dose intensity could be further escalated to 2.33 compared with our standard regimen by decreasing the interval between treatment cycles to 2 weeks. However, at this dose intensity less than a third of patients received their planned treatment on time. All the delays were due to thrombocytopenia.

Adult↗
PubMed1996

Evaluation of cytogenetic conversion to Ph- haemopoiesis in long-term bone marrow culture for patients with chronic myeloid leukaemia on conventional hydroxyurea therapy, on pulse high-dose hydroxyurea and on interferon-alpha.

Long-term bone marrow culture (LTBMC) has been used successfully in autologous transplantation in chronic myeloid leukaemia (CML). However, variation between patients in the recovery of Ph- cells in culture limits the application of this procedure to a minority. Treatment that effectively reduces in vivo tumour burden prior to initiation of LTBMC may improve the selection of Ph- cells in culture. To test this hypothesis we evaluated the frequency and degree of cytogenetic conversion to Ph- haemopoiesis in LTBMC from four independent groups of CML patients: Untreated (n = 19); conventional dosage of hydroxyurea (HU) (n = 10); pulse high-dose HU (P-HU) (n = 22) and interferon (IFN)-alpha (n = 12). In this study IFN-alpha therapy resulted in a significantly higher incidence of patients with detectable Ph- clonogenic cells in the marrow (P = 0.01) and with > or = 50% Ph- haemopoiesis in LTBMC as compared to newly diagnosed patients (P = 0.05). Also, sequential culture studies undertaken in 14 CML patients at diagnosis and following the start of pulse highdose HU therapy showed that in eight patients the average proportion of Ph- metaphases detected in LTBMC substantially increased from 1.7% (range 0-7) at diagnosis to levels of 71% (range 14-100) after treatment. Therefore we conclude that the use of IFN or pulse high-dose HU in early stage disease appears to create an opportunity to harvest the marrow for long-term culture (LTC) purging with reduced leukaaemic burden.

Adult↗
PubMed1996

The relative spatial distribution of in vitro-CFCs in the bone marrow, responding to specific growth factors.

Haemopoietic progenitor cells are stimulated by a range of growth factors which promote colony growth in culture. The progenitors are a part of an age-structured developmental hierarchy in the tissue. The growth factors, although overlapping in their effects, stimulate cells preferentially at different stages in this programme. Femoral bone marrow was fractionated into axial (close to the central venous sinus) and marginal (close to the bone surface) cells. Progenitors which responded to IL-3, GM-CSF, G-CSF, M-CSF and SCF were then assayed in soft agar cultures. Consequent plots of their spatial distributions showed that the more primitive cells in vitro (responding to IL-3) were concentrated close to the bone surface. The peak concentrations of cells responding primarily to growth factors with progressively more affinity to more mature progenitor cells correspondingly appeared progressively further from the bone surface and closer to the point of release at the central venous sinus. This suggests that the developmental/maturational process in haemopoiesis is accompanied by a progressive movement of cells from the bone surface towards the central axial regions of the bone cavities. The most primitive cells are however exposed, close to the centre of the cavity, by a combination of SCF and G-CSF (or by a 50-fold increase in G-CSF concentration alone). These results corroborate earlier data which indicate a developmental movement of cells from the centre of the marrow tissue towards the bone surface and back again, sequentially encountering a series of growth factors which promote their differentiation into mature cells, for release at the central venous sinus.

Animals↗
PubMed1996

Peripheral blood progenitor cell transplantation: where do we stand? Chairman's Summary of the European School of Oncology Task Force meeting Peripheral Blood progenitor cell's held September 29-30, 1995.

Whether or not peripheral stem cells have an unlimited capacity for self renewal is debated. However, everyday haematopoietic requirements are met by progenitors; and it seems that few "real' stem cells are needed. Although we may not yet have identified these "true' stem cells, for practical purposes the long term culture-initiating cells (LTC-ICs) are a close approximation. To date, experience in peripheral blood progenitor cell (PBPC) transplantation is largely confined to non-ablative regimens. It is therefore difficult to determine the number of PBPCs needed to effect long-term reconstitution. The number of tumour cells present among mobilised PBPCs can be reduced using the CD34 affinity column and by positive purging methods. The ex vivo expansion of CD34 cells also has the effect of diluting tumour cell concentration. In clinical use, PBPC transplantation has a proven role in support of high dose chemotherapy in certain haematological and oncological malignancies but the concept of dose intensification is not universally accepted. With the exception of leukaemia, lymphoma, myeloma or relapsed testicular cancer and possibly some subgroups of breast cancer; high dose chemotherapy does not demonstrate a survival benefit. For patients with CML, autografting with Ph- cells appears to become a useful alternative to allogeneic BMT. Allogeneic PBPC transplantation may have potential, though work is preliminary. Cord blood transplantation between matched siblings is viable, but it is not yet clear whether this source will increase the donor pool for adults needing allogeneic transplantation. For gene therapy using haematopoietic cells to be effective, a greatly increased rate of transduction will be needed. Meeting in Paris in September 1995, a European School of Oncology Task Force considered a number of important questions relating to peripheral blood progenitor cell (PBPC) physiology and transplantation. This review is a brief account of their conclusions.

Antineoplastic Agents↗
PubMed1996

Mobilisation kinetics of primitive haemopoietic cells following G-CSF with or without chemotherapy for advanced breast cancer.

BACKGROUND: The objective of this study was to determine the optimal conditions for blood progenitor cell harvest for transplantation, with main emphasis on the mobilisation kinetics of primitive, marrow repopulating cells. PATIENTS AND METHODS: Sixteen patients with advanced breast cancer were treated with 4 cycles of dose escalating FAC chemotherapy (5-fluorouracil, adriamycin, cyclophosphamide) each followed by 10 micrograms/kg/d G-CSF for 13 days. We assessed the number of colony-forming cells (CFC), and estimated the long-term culture initiating cells (LTC-IC) and CD34+ cells during the recovery phase of cycle 1 and 4 of chemotherapy, and during additional periods of G-CSF administration either preceding or following the full course of chemotherapy. RESULTS: The highest peak numbers of CFC per ml of blood (median 10489, range 860-39282) were mobilised after the first cycle of chemotherapy. The lowest peak numbers of CFC were obtained during the recovery phase from cycle 4 (median 4739, range 40-26789). In contrast, the numbers of CD34+ cells per ml of blood were significantly higher in cycle 4 (median 650, range 30-2600 x 10(2)) compared to those of cycle 1 (median 240, range 20-770 x 10(2)). The peak numbers of CFC mobilised by G-CSF before commencement and after the cessation of chemotherapy were equivalent, with a median of 5470 (range 1056-25669) and 5948 (range 2710-38975) per ml of blood, respectively. However, while mononuclear cells (MNC) collected at the days of maximal CFC mobilization following G-CSF administration before or after cycle 1 were similar to normal bone marrow MNCs in their ability to generate haemopoiesis when seeded onto performed irradiated stroma, those collected after cycle 4 or during G-CSF administration after the cessation of chemotherapy were markedly compromised in this respect. CONCLUSIONS: Our results indicate that repeated cycles of FAC chemotherapy followed by G-CSF result in a far lower number of LTC-IC than of CFC mobilised into the circulation. Furthermore although the combination of chemotherapy and G-CSF mobilised the highest numbers if CFC, G-CSF alone pre-chemotherapy was more effective at mobilising LTC-IC. These data indicate that neither the numbers of CFC mobilised nor the numbers of CD34+ cells are necessarily a reliable indicator for the putative marrow repopulating capability of the blood cells mobilised with chemotherapy plus G-CSF.

Adult↗