Modified chemotherapy with carmustine, cytarabine, cyclophosphamide, and 6-thioguanine (BACT) and autologous bone marrow transplantation in 24 poor-risk patients with acute lymphoblastic leukemia.

Twenty-four poor-risk patients with acute lymphoblastic leukemia received a modified regimen of carmustine, cytarabine, cyclophosphamide, and 6-thioguanine (BACT) followed by autologous bone marrow transplantation (ABMT). Nineteen patients were in second or subsequent complete remission (CR) when treated with this regimen; 3 died early, 2 died of pneumonia in CR, 11 relapsed within 3 months (median), and 3 remain in CR with no maintenance therapy 14-24 months after ABMT. Of the 5 patients with measurable disease who were treated, 3 had CR and 1 remains in CR without maintenance therapy more than 28 months after ABMT. The toxicity of this regimen was acceptable, but late pulmonary toxic effects remain a major concern. These results are poor in terms of efficacy, and new effective methods of eradicating acute lymphoblastic leukemia in patients with poor prognosis should be investigated.

High-dose busulfan and cyclophosphamide with autologous bone marrow transplantation support in advanced malignancies in children: a phase II study.

Twenty children with advanced, nonleukemic malignancies entered a phase II study of high-dose busulfan-cyclophosphamide followed by bone marrow transplantation (BMT). All had disease refractory to conventional and/or high-dose chemotherapy (HDC). There were ten neuroblastoma patients, six non-Hodgkin's lymphoma, three Ewing's sarcoma, and one rhabdomyosarcoma. Eight had primarily resistant disease, ten were in second progressive relapse, and two in third progressive relapse. One patient was not evaluable for response. Among the 19 evaluable patients the responses observed were complete response (CR), seven; partial response (PR), three; objective effect, five; and failure, four. However, survival was poor: 15 patients died, two are alive with disease, and three are alive with no evidence of disease (NED) at 8+, 11+, 14+ months post-BMT. Toxicity was high but considered as acceptable, taking into account the terminal state of these patients. Seven treatment-related deaths were observed. This combination therapy proved to be highly effective, with a response rate of 50%, and its value for eradication of residual disease in less advanced patients should be investigated.

High-dose polychemotherapy with autologous bone marrow transplantation in children with relapsed lymphomas.

Sixteen children with non-Hodgkin's lymphoma (NHL) who had relapsed were treated with high-dose chemotherapy with BCNU, cyclophosphamide, cytarabine, 6-thioguanine (high-dose chemotherapy [HDC]) and autologous bone marrow transplantation (ABMT). Eleven complete responses were obtained and five patients remain in prolonged complete unmaintained remission 77+ to 152+ weeks after treatment. The best results were obtained in patients with CNS involvement and when this regimen was used after complete remission or partial response was obtained by other means. The results appear to be better in B-cell than in T-cell lymphomas, but the numbers are too small for statistical assessment. The use of ABMT rendered the pancytopenic period short and safe, despite the use of drug doses higher than those previously described for this HDC. The frequency of interstitial pneumonitis, possibly related to pulmonary toxicity of chemotherapy, remains a major concern. These results show that this regimen can help to cure some patients but its toxicity prohibits its use in primary therapy.

Prevention of graft-versus-host disease in HLA-matched bone marrow transplantation for malignant diseases: a multicentric study of 62 patients using 3-pan-T monoclonal antibodies and rabbit complement.

In order to evaluate the effectiveness and reproducibility of T cell depletion in human leukocyte antigen (HLA)-matched bone marrow graft to prevent graft-v-host disease (GVHD), our multicentric study (nine different centers) investigated 62 consecutive patients with poor prognosis leukemia or hematosarcoma from June 1984 to November 1985. The data were updated October 1, 1986, and the mean follow-up was 18 +/- 4.3 months. T cells were depleted with a combination of 3-pan-T cell monoclonal antibodies (CD2 "D66"; CD5 "A50"; CD7 "I21") with a single incubation of rabbit complement (C'). The average number of T cells infused was 0.66 X 10(6) +/- 0.56/kg body weight. Twenty-six patients received chemoprophylaxis for GVHD, 16 received methotrexate, and ten received cyclosporin A. Only a single case of severe (greater than grade II) GVHD was observed, yet the incidence of graft failure was 19%. Factors that might have influenced the occurrence of graft failure appear to be the lack of radiotherapy in the conditioning regimen; the conditioning regimen itself (fractionated total body irradiation [TBI], 12 Gy, v single dose is better than TBI, 10 Gy, but still not statistically significant); and the age of the patients (high-risk after 30 years of age). In contrast, neither the number of nucleated cells reinfused nor the level of T cell depletion (provided the T cells were below critical numbers) seemed to have an influence, nor did chemoprophylaxis for GVHD or splenectomy in chronic granulocytic leukemia (CGL) patients. The survival of graft failure patients was very poor (one of 11; survival at 15 months of the initial graft). Thus, our study demonstrates the reproducibility and high effectiveness in preventing GVHD by immunodepletion of T cells in a large-scale multicentric assay, in which compliance with the protocol of immunodepletion was reasonably good. This study thus provides interesting clues to overcoming graft rejection.

Repeated high-dose chemotherapy followed by purged autologous bone marrow transplantation as consolidation therapy in metastatic neuroblastoma.

Among 62 children over 1 year of age at diagnosis, who were treated for stage IV neuroblastoma, 33 entered complete remission (CR) or good partial remission (GPR) after conventional therapy and received high-dose chemotherapy (HDC) with in vitro purged autologous bone marrow transplantation (ABMT) as consolidation therapy. The HDC was a combination of carmustine (BCNU), teniposide (VM-26), and melphalan. Thirty-three patients received one course of this regimen, and 18 received two courses. At present, 16 of the 33 grafted patients are alive in continuous CR, with a median follow-up of 28 months. Toxicity of this regimen was tolerable, principally marked by bone marrow depression and gastrointestinal (GI) tract complications. Four complication-related deaths were observed. Relapse post-ABMT occurred most often in the bone marrow. Under this treatment, actuarial disease-free survival is improved compared with that observed under conventional therapy.

Percutaneous autologous bone-marrow grafting for nonunions. Influence of the number and concentration of progenitor cells.

BACKGROUND: Bone marrow aspirated from the iliac crest contains progenitor cells that can be used to obtain bone-healing of nonunions. However, there is little available information regarding the number and concentration of these cells that are necessary to obtain bone repair. The purpose of this study was to evaluate the number and concentration of progenitor cells that were transplanted for the treatment of nonunion, the callus volume obtained after the transplantation, and the clinical healing rate. METHODS: Marrow was aspirated from both anterior iliac crests, concentrated on a cell separator, and then injected into sixty noninfected atrophic nonunions of the tibia. Each nonunion received a relatively constant volume of 20 cm(3) of concentrated bone marrow. The number of progenitor cells that was transplanted was estimated by counting the fibroblast colony-forming units. The volume of mineralized bone formation was determined by comparing preoperative computerized tomography scans with scans performed four months following the injection. RESULTS: The aspirates contained an average (and standard deviation) of 612 +/- 134 progenitors/cm(3) (range, 12 to 1224 progenitors/cm(3)) before concentration and an average of 2579 +/- 1121 progenitors/cm(3) (range, 60 to 6120 progenitors/cm(3)) after concentration. An average total of 51 x 10(3) fibroblast colony-forming units was injected into each nonunion. Bone union was obtained in fifty-three patients, and the bone marrow that had been injected into the nonunions of those patients contained >1500 progenitors/cm(3) and an average total of 54,962 +/- 17,431 progenitors. The concentration (634 +/- 187 progenitors/cm(3)) and the total number (19,324 +/- 6843) of progenitors injected into the nonunion sites of the seven patients in whom bone union was not obtained were both significantly lower (p = 0.001 and p < 0.01, respectively) than those in the patients who obtained bone union. The volume of the mineralized callus measured at four months on the computerized tomography scans of the patients who had union ranged from 0.8 to 5.3 cm(3) (mean, 3.1 cm(3)). There was a positive correlation between the volume of mineralized callus at four months and the number (p = 0.04) and concentration (p = 0.01) of fibroblast colony-forming units in the graft. There was a negative correlation between the time needed to obtain union and the concentration of fibroblast colony-forming units in the graft (p = 0.04). CONCLUSIONS: Percutaneous autologous bone-marrow grafting is an effective and safe method for the treatment of an atrophic tibial diaphyseal nonunion. However, its efficacy appears to be related to the number of progenitors in the graft, and the number of progenitors available in bone marrow aspirated from the iliac crest appears to be less than optimal in the absence of concentration.

[The "forgotten ones of reperfusion" in acute myocardial infarction: lessons from the ESTIM Limousin registry]. / Les "oubliés de la reperfusion" dans l'infarctus du myocarde aigu. Leçons du registre ESTIM limousin.

The management of acute coronary syndrome (ACS) with ST elevation in daily practice does not always comply with the official guidelines. In effect, many patients do not benefit from coronary recanalisation despite being eligible. They could be described as the 'reperfusion forgotten ones'. The Limousin ESTIM study allowed us to evaluate their numbers and characteristics between 2001 and 2003. Between 1 June 2001 and 31 December 2003, 958 patients with ST+ ACS were managed within 24 hours. Among this cohort, 47% of patients did not benefit from reperfusion treatment with fibrinolysis or angioplasty. In spite of early management, the rate of non-reperfusion was significant: 30% before the third hour, and 50% between 3 and 6 hours. With univariate and multivariate analysis, the predictive features for non-reperfusion were age, length of time between onset of pain and presentation, type of admission route, absence of a call to the emergency ambulance service, and the characteristics of the ECG tracing. These data have prompted education and training, adapted for specific regions. Despite some significant improvements, the rate of non-perfusion in 2004 still remains 35% in the first 24 hours, comparable with figures in the recent literature. Being aware of this problem, taking specific action and continued evaluation with surveys like this remain important.

Clonal T-cell colony formation in agar culture: an attractive assay to test the T-cell depletion from bone marrow.

Current studies suggest that the depletion of T-lymphocytes from donor marrow is an effective method for preventing acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation in man. To deplete the T-lymphocytes from bone marrow cells we use either monoclonal anti-T-cell antibodies and complement or T101 ricin A-chain immunotoxin. Residual T-lymphocytes are analyzed by their capacity to form clonal T-cell colonies in the presence of phytohemagglutinin (PHA), accessory cells, and recombinant interleukin 2. The method is compared to immediate indirect immunofluorescence (iF) and thymidine incorporation by marrow cells stimulated by PHA. IF is not suitable for evaluating the depletion by immunotoxin, and the interpretation of thymidine incorporation is generally questionable. The results of the colony formation show that the sensitivity of the colony assay is close to that of iF when T cells are depleted by complement lysis, and the sensitivity of the colony assay is not dependent upon the depletion procedure. Therefore, the T-cell colony assay is a simple functional control for the quality of bone marrow T-cell depletion, especially for T-cell depletion by immunotoxin.

T-cell depletion of bone marrow transplants: assessment of standard immunological methods of quantification.

The efficacy of bone marrow transplant (BMT) T-cell depletion for the prevention of acute graft-versus-host disease (GVHD) has been demonstrated in animal models and in clinical studies. The importance of T-cell depletion has to be evaluated with standardized methods suitable for routine purposes. We report herein an in vitro mature T-cell depletion using a cocktail of three monoclonal antibodies (CD2, CD5, and CD7) and baby rabbit complement in 38 histocompatibility leucocyte antigen (HLA)-identical BMT with no more than grade II acute GVHD. The T-cell depletion was quantified using three prestandardized immunological methods: immunofluorescence (IF) analysis, SRBC-rosetting assay, and PHA proliferation assay. A mean of 97.5% IF-assessed T-cell depletion was achieved in the 38 BMT. The immediate IF analysis using three distinct sets of anti-T-cell monoclonal antibodies allowed us to detect a mean of 1.2% residual T cells. The SRBC-rosetting assay was not useful to quantify T-cell depletion because no residual SRBC-rosette-forming cells could be detected in every case. The results of a prestandardized PHA-induced proliferation assay gave a mean 96.7% inhibition of proliferation, and they were correlated with the IF results although the IF threshold of detection was higher. From these data we conclude that our in vitro T-cell-depletion procedure is reproducible and that standardized simple immunological methods such as immediate immunofluorescence analysis and PHA proliferation assay provide good tools to assess a T-cell depletion effective in the prevention of acute GVHD.

Stage IV neuroblastoma in patients over 1 year of age at diagnosis: consolidation of poor responders with combined busulfan, cyclophosphamide and melphalan followed by in vitro mafosfamide-purged autologous bone marrow transplantation.

In an attempt to improve the poor prognosis of poor responders with stage IV neuroblastoma, a new combined high-dose chemotherapy conditioning regimen was tested. Event-free and overall survival, as well as the incidence of complications, were analysed. Twenty-five children aged 12-146 months at diagnosis entered this study. All were in complete remission (CR) at the time of high-dose chemotherapy. Two or three different protocols had been necessary for them to achieve a CR. High-dose chemotherapy consisted of a combination of busulfan (600 mg/m2), cyclophosphamide (4400 mg/m2) and melphalan (140 mg/m2). It was followed by autologous bone marrow transplantation (ABMT). The bone marrow graft was purged in vitro with mafosfamide. The probability of event-free survival (EFS) at 5 years post-ABMT was 34%, compared to < 8% in a historical series. Toxicity was severe but manageable and 2 complication-related deaths were observed. Veno-occlusive disease was the most frequent extrahaematopoietic complication encountered, but its outcome was always favourable. By using a very intensive conditioning regimen consisting of a combination of three alkylating agents, the EFS of poor responders with metastatic neuroblastoma was improved and similar to that of good responders. When compared with a previously published similar series of patients, the improvement in survival appears probably related to intensification of the conditioning regimen.

Long-term survivors after salvage high dose chemotherapy with bone marrow rescue in refractory germ cell cancer.

Between April 1984 and May 1985, 17 heavily pretreated patients with relapsing or refractory germ cell tumours were treated with cisplatin 40 mg/m2/day, days 1-5; etoposide 350 mg/m2/day, days 1-5; cyclophosphamide 1600 mg/m2/day, days 2-5 and autologous bone marrow transplantation on day 8 as consolidation of conventional salvage chemotherapy. None of the 11 refractory patients and 4 of the 6 responders to prior salvage treatment are long-term survivors at 68, 72, 74 and 74 months. Mean aplasia duration was 17 days and there were 7 documented episodes of septicaemia in 17 febrile patients. 1 patient died of treatment. Among the 4 survivors, 2 patients have a sustained grade II invalidating neuropathy. We conclude that this regimen is not recommended as salvage therapy in refractory patients but may be a useful consolidation treatment in patients responding to conventional salvage chemotherapy.

High-dose chemotherapy with etoposide, cyclophosphamide and escalating dose of carboplatin followed by autologous bone marrow transplantation in cancer patients. A pilot study.

25 patients with poor-prognosis malignancies were treated with a combination of fixed-dose etoposide (1750 mg/m2), cyclophosphamide (6400 mg/m2) and escalating doses of carboplatin (from 800 to 1600 mg/m2) followed by autologous bone marrow transplantation (ABMT). The median duration of granulocytopenia (< 500/mm3) and thrombocytopenia (< 20,000/mm3) was 23 days and 20.5 days, respectively. The main non-haematological toxicity was gastro-intestinal, with moderate to severe diarrhoea in 15 patients. No significant renal toxicity was observed. 2 patients died early due to toxicity. The overall response rate was 58% including 42% having complete responses. 4 of the 25 patients are alive with no evidence of disease at 22, 27, 40 and 43 months after ABMT. The encouraging antitumoral activity of this regimen makes it a good candidate for intensified chemotherapy in patients with various malignancies. Toxicity is acceptable and may be reduced in the near future with the widespread use of haematopoietic growth factors.

Failure of a CD18/anti-LFA1 monoclonal antibody infusion to prevent graft rejection in leukemic patients receiving T-depleted allogeneic bone marrow transplantation.

CD18 antibodies react with the common beta chain of the human leukocyte function antigen (LFA1)* and thus block the functions mediated by the three identified molecules in humans. A murine CD18 monoclonal antibody was infused in 8 leukemic patients receiving allogeneic T-depleted bone marrow transplantation in order to prevent graft rejection. This was part of the conditioning, including total-body irradiation and high-dose chemotherapy, given to all patients. To prevent graft-versus-host disease the donor bone marrow T cells were depleted using complement-mediated cytolysis or a ricin A conjugate immunotoxin, and cyclosporine or methotrexate were given posttransplant. A persistent level of free circulating anti-LFA1 antibody was detected in 5/8 patients. Despite this, 5 graft failures occurred, with 2 patients experiencing late rejection (days 60 and 97) following HLA-identical transplantation and 3 patients having no engraftment following haplo-mismatched transplant. One other patient died of early sepsis. Only 2 patients (who differed at 1 HLA locus from their donor) are alive with long-term complete chimerism (300 and 315 days). Transient inhibition of recipients' leukocyte functions with an anti-LFA1 antibody did not appear to facilitate engraftment of allogeneic T-depleted marrow transplantation for leukemias.

Nonbacterial nonfungal interstitial pneumonitis following autologous bone marrow transplantation in children treated with high-dose chemotherapy without total-body irradiation.

Between February 1979 and May 1986, 165 children were treated with autologous bone marrow transplantation after high-dose chemotherapy without total-body irradiation for a hematologic malignancy or a solid tumor. Nonbacterial nonfungal interstitial pneumonitis was observed in 24 children and was the cause of death in 11 cases. Of the 24 cases of interstitial pneumonitis, 14 were considered to be idiopathic. Cytomegalovirus was the major pathogenic agent detected in the 10 other cases of interstitial pneumonitis (n = 5), followed by Herpes zoster (n = 2), Pneumocystis carinii (n = 1), tumor (n = 1), and adenovirus (n = 1). The only factor found to correlate significantly with the increased rate of interstitial pneumonitis was the use of high-dose 1-3 bis chloroethyl-1 nitrosourea (BCNU) (600 mg/m2), whereas BCNU at a dose of 300 mg/m2 did not affect this rate. These data, when compared with the literature, show a lower incidence of interstitial pneumonitis than in allogeneic transplantations, and an incidence similar to that observed in syngeneic transplantations, although there was no radiation toxicity in this series.

Predictive value of host-specific donor helper T-cell precursor frequency for acute graft-versus-host disease and relapse in HLA-identical siblings receiving allogeneic bone marrow transplantation for hematological malignancies.

BACKGROUND: Acute graft-versus-host disease (aGVHD) is still one of the main causes of morbidity and mortality after allogeneic bone marrow transplantation. Attempts to avoid GVHD are associated with an increased risk of relapse, probably because the graft-versus-leukemia effect is also abrogated. It was recently suggested that a high frequency of host-specific donor helper T cell precursors (HTLp) might be predictive of significant aGVHD (grade > or = II). METHODS: We retrospectively studied the frequency of HTLp by means of simplified limiting-dilution analysis to determine its predictive value for aGVHD and relapse. Pre-bone marrow transplantation, host-specific donor HLTp frequencies were analyzed in 32 patients who had received marrow from HLA-identical siblings for hematological malignancies, in terms of aGVHD and relapse. RESULTS: HTLp frequencies were significantly higher in patients who had aGVHD > or = grade II (n=14) than in those without aGVHD (n=18) (P=0.007). Patients who relapsed (n=13) had significantly lower HTLp frequencies than those who did not relapse (n=19) (P<0.0001). The probabilities of relapse (Kaplan-Meier method) when the HTLp frequency was higher and lower than 1/200,000 were 0% and 88%, respectively (P<0.0001). CONCLUSIONS: The definition of HTLp cut-off values predictive of aGVHD and relapse should contribute to donor selection and could open the way to protocols adapting immunomodulation to the likely risk of aGVHD and relapse.

Enhanced in vitro cytotoxicity of 1,3-bis-(2-chloroethyl)-1-nitrosourea or buthionine sulfoximine combined with a reactive oxygen-generating enzyme immunotoxin.

The glutathione inhibitor drugs, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and buthionine sulfoximine (BSO), were tested in vitro in order to assess their cytotoxic effectiveness when combined with an enzyme immunotoxin (eIT) composed of a T-cell reactive monoclonal antibody (mAb) 097 coupled to the reactive oxygen-generating enzyme, glucose oxidase (GO) (EC 1.1.3.4). As targets of this eIT we used mature human T-cells or leukemia cells that expressed the 097 epitope. We found that treatment of the cells with subtoxic amounts of mixtures of both a drug and the 097 eIT markedly potentiated cytotoxicity compared to either drug or eIT alone.

Effects of irradiation on human platelets stored at 20 degrees C.

The morphologic and functional properties of platelets after irradiation with 2500 rads and storage, in first-generation containers, for 48 h in a liquid phase at 20 degrees C with continuous horizontal agitation have been analyzed and compared with a control group of the same platelets which were not irradiated. The preservation technique induced changes in the morphology and aggregation stimulated by ADP and collagen. However, no significant differences were found between the irradiated and non-irradiated groups. Irradiation is not a conditioning factor to add to the hazards of preserving platelets in a liquid phase.

Rapid technique for cryopreservation of Echinococcus multilocularis metacestodes.

Cysts of E. multilocularis were minced to prepare a crude homogenate and after addition of glycerol at a final concentration of 10%, cryopreservation was performed at a rate of 1 degree C min-1 in a controlled-rate freezer. The aliquots were subsequently stored in liquid nitrogen. All 22 isolates tested were successfully cryopreserved and their viability maintained.

Effects of cryopreservation on the proliferation and anticoagulant activity of human saphenous vein endothelial cells.

Human saphenous veins were cryopreserved in 4% human albumin and 10% dimethyl sulfoxide. The effect of cryopreservation on endothelial cells was studied in terms of the anticoagulant activity of thrombomodulin and in terms of cell proliferation. After storage for 2 weeks at -150 degrees C, 0.45 +/- 0.07 x 10(5) endothelial cells/cm2 were detected in cryopreserved veins and 1.03 +/- 0.04 x 10(5) endothelial cells/cm2 in fresh veins (p < 0.01). The thrombin-catalyzed activation of protein C decreased after cryopreservation, indicating altered thrombomodulin activity in the endothelial cells. On a cell number basis, the release of soluble thrombomodulin was three times higher from the cryopreserved endothelium than from the fresh endothelium (p < 0.05). The amount of spontaneous release of von Willebrand factor from the endothelial surface was not significantly different between fresh and cryopreserved veins. Endothelial cells were cultured from fresh veins and from their cryopreserved counterparts. On plating of endothelial cells in primary culture, the number of adhered cells was 0.9 +/- 0.09 x 10(3) cells/cm2 from fresh veins and 0.25 +/- 0.03 x 10(3) cells/cm2 from cryopreserved veins (p < 0.01). The positive immunohistochemical stain for von Willebrand factor indicated that the endothelial cell character was maintained after cryopreservation. The endothelial desquamation with loss of anticoagulant function and the slow proliferation of surviving cells in vitro suggest an impaired endothelial healing in vivo. The loss of anticoagulant activity complicates the problems of the exposure of thrombogenic subendothelial matrix to blood in implanted cryopreserved veins.

Hemopoietic reconstitution after repeated autologous transplantation with mafosfamide-purged marrow.

Twenty-nine children (median age: 41 months) with advanced solid tumors received, as consolidation therapy, two consecutive courses of high-dose chemotherapy (HDC) followed by mafosfamide-purged autologous marrow transplantation (ABMT) with a 3- to 4-month interval between each course. The malignancies were neuroblastoma (n = 22), Ewing's sarcoma (n = 5) and rhabdomyosarcoma (n = 2). Patients received a preparatory regimen consisting of combined high-dose melphalan before each ABMT, with the exception of five patients who received busulfan and cyclophosphamide before the second ABMT. Prior to HDC, bone marrow sufficient for two transplantations was harvested in remission, treated with mafosfamide (50 micrograms/ml) and cryopreserved. Following incubation with the drug, a consistent inhibition (greater than 99%) of granulocyte and macrophage colony-forming units was observed. Despite the elimination of measurable hematopoietic progenitors, all patients underwent engraftment within a similar period of time after the first and the second ABMT. However, peripheral leukocyte and granulocyte recovery was delayed (median 26 and 28 days, respectively, after the first graft; median 27 and 28 days after the second graft). No difference was observed in the bacterial infections following the first and second ABMT. One patient died after the second transplant with diffuse aspergillosis. Recovery to 50 x 10(9) platelets/l occurred after a median 42 days (range 19-71) after the first ABMT and 43 days (range 14-110) after the second. Two patients died of recurrent disease before attaining a normal platelet level. One patient remained thrombocytopenic and died from visceral failure at day 200. These results demonstrate the feasibility of repeated ABMT with mafosfamide-treated marrow.(ABSTRACT TRUNCATED AT 250 WORDS)