Preclinical modeling of myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) represent a heterogeneous group of hematological clonal disorders. Here, we have tested the bone marrow (BM) cells from 38 MDS patients covering all risk groups in two immunodeficient mouse models: NSG and NSG-S. Our data show comparable level of engraftment in both models. The level of engraftment was patient specific with no correlation to any specific MDS risk group. Furthermore, the co-injection of mesenchymal stromal cells (MSCs) did not improve the level of engraftment. Finally, we have developed an in vitro two-dimensional co-culture system as an alternative tool to in vivo. Using our in vitro system, we have been able to co-culture CD34+ cells from MDS patient BM on auto- and/or allogeneic MSCs over 4 weeks with a fold expansion of up to 600 times. More importantly, these expanded cells conserved their MDS clonal architecture as well as genomic integrity.

Phase I study of high-dose topotecan with haematopoietic stem cell support in the treatment of ovarian carcinomas: the ITOV 01 protocol.

Topotecan has demonstrated activity in ovarian carcinomas. In order to increase the tumour response rate and to define the maximum tolerated dose (MTD) of topotecan, we decided to develop a high-dose phase I regimen supported by stem cell support. High-doses schedules using a 1-day single administration have MTDs of 10.5 (24 h continuous infusion (CI)) or 22.5 mg/m2 (30 min infusion). Five-day CI induces grade IV mucositis at high doses (MTD<12 mg/m2). We chose to administer topotecan in a 5-day schedule with a 30 min daily infusion. Patients were scheduled to receive one cycle of therapy. The first dose level was 4.0 mg/m2/day x 5 days. Limiting toxicities were defined as toxic death, grade IV non-haematopoietic or haematopoietic toxicity >6 weeks. From August 1998 to April 2002, 49 patients were included. Forty-three patients have completed one course and 15 have received two cycles. One patient treated at level 7 mg/m2/day died of sepsis. Median duration of grade IV neutropenia was 9 days. Two episodes of grade IV diarrhoea were observed at level 9.5 mg/m2/day. Pharmacokinetic data were linear within the dose range of 4-9.0 mg/m2/day. The MTD was reached at 9 mg/m2/day x 5 days.

Clinical phase I and pharmacokinetic study of S 16020, a new olivacine derivative: report on three infusion schedules.

S 16020, a new 9-OH olivacine derivative, is a novel topoisomerase II inhibitor with activity in cell lines presenting the classical multidrug resistance phenotype. This report summarizes, in addition to pharmacokinetic data, the whole phase I clinical experience of S 16020 using three different infusion schedules. Asthenia and skin toxicity were the main side effects. In an attempt to understand the skin toxicity mechanism, experiments in animals were performed, the results of which are reported. S 16020 showed rapid tumor necrotizing activity in some patients, with soft tissue metastases of epidermoïd tumors and pain at the tumor site. To document the side effects of S 16020 and tumor site reactions (pain, edema, inflammatory signs), inflammatory parameters and some cytokines were measured. In our patients there was no hemolysis and no detection of anti-S 16020 antibodies, confirming the absence of immunogenicity of the compound. Based on the overall data of the three infusion schedules of S 16020, the dose of 100 mg/m(2) over 3 h every 3 weeks was selected for phase II studies.

Self-healing generalized infantile myofibromatosis with elevated urinary bFGF.

We report a case of generalized infantile myofibromatosis with favorable outcome despite systemic involvement. Elevated urinary bFGF levels during the active phase of the disease suggested an angiogenic stimulation in the pathogenesis of myofibromatosis.

Study of cord blood natural killer cell suppressor activity.

We tested the immunosuppressive effect of cord blood (CB) natural killer (NK) cells using highly purified CB NK cells in mixed lymphocyte cultures (MLC) containing autologous CB T cells as responders. Control cultures were done without NK cells. Our findings revealed that CB NK cells induced a dose-dependent inhibition of T lymphocyte proliferation as evidenced by decreased 3H-thymidine incorporation in MLC. The T cell alloproliferation was significantly decreased in the presence of an NK cell to responder cell ratio of 0.1, 0.2 or 0.4 compared with control cultures done without NK cells (p=0.02, 0.003 and 0.0002, respectively). T lymphocyte inhibition was also achieved using irradiated CB NK cells and still demonstrable on addition of disparate CB NK and T cells to the MLC. In agreement with previous reports, adult blood NK cells inhibited the alloreactive T cells in the MLC using adult T lymphocytes as responders. Compared to control cultures done without NK cells, statistically significant inhibition of 3H-thymidine incorporation in MLC was observed at a ratio of NK cells to responder cells ratio of 0.2 or 0.4 (p=0.02). To investigate the mechanism whereby CB NK cells can interfere with the development of alloreactive T cells in MLC, we measured the tumour necrosis factor-alpha (TNF-alpha) concentrations in MLC supernatants using NK cell-depleted or unseparated CB mononuclear cells (MNC) as responders. The results revealed significantly high levels of TNF-alpha in the absence of NK cells (p=0.007). We conclude that CB NK cells suppress alloreactive T lymphocytes as do their counterparts in adult blood. However, the high NK to T cell ratio in CB could contribute to a more marked suppressive potential compared to that in adult blood. The mechanism of NK-mediated inhibition is likely related to disruption of the TNF-alpha pathway of T-lymphocyte activation.

Are basic fibroblast growth factor and vascular endothelial growth factor prognostic indicators in pediatric patients with malignant solid tumors?

Angiogenesis plays an important role in the growth, progression, and metastasis of solid tumors. Among angiogenic factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) appear to be useful markers in adults with cancer. The aim of this pilot study was to determine the levels of VEGF in serum and bFGF in serum and urine of children with solid tumor at diagnosis (as measured by ELISA), and to investigate whether these parameters provide prognostic information. Forty consecutive patients with different types of cancer were prospectively included in this study. Median values of all studied angiogenic factors were higher in patients than in controls (n = 40), and the differences were statistically significant for bFGF in serum and urine: 10 versus 3 pg/ml (P = 0.0004) and 6406 versus 0 pg/g of creatinine (P < 0.0001), respectively. Among patients, median serum values of bFGF and VEGF were higher in children with metastatic disease (n = 14) than in those with localized disease (n = 26). The difference was statistically significant for serum bFGF: 17.5 versus 6 pg/ml (P = 0.02). Serum angiogenic factor levels correlated with outcome. The estimated event-free survival at 3 years was 79% for patients with normal bFGF values (n = 13) versus 42% (n = 26; P = 0.02) for those with high levels, and 71% in case of normal VEGF values (n = 20) versus 38% (n = 19; P = 0.04) for those with high levels. No benefit of normal urinary bFGF values was observed. Our results provide a rationale for exploring the clinical interest of bFGF and VEGF measurements in body fluids of a larger group of children with cancer.

Cytokines and vascular cell adhesion molecule-1 in the blood of patients undergoing HPC mobilization.

BACKGROUND: The mechanism of HPC mobilization in humans is unclear. In this study, the relationship between PBPC mobilization and blood levels of G-CSF, endogenous cytokines (IL-8, SCF, thrombopoietin [TPO]), and the vascular cell adhesion molecule-1 (VCAM-1) was analyzed in patients with malignancy who were undergoing a PBPC mobilization regimen. STUDY DESIGN AND METHODS: Fifty-four patients with multiple myeloma (MM) and 29 with breast cancer (BC) underwent a mobilization regimen combining conventional chemotherapy and G-CSF up to the last day of PBPC collection. The CD34+ cell count was determined on each day when leukapheresis was scheduled. Venous blood samples (n = 117) were drawn before apheresis for CD34+ cell count (flow cytometry) and cytokine (G-CSF, IL-8, SCF, TPO) and VCAM-1 measurements (ELISA). RESULTS: In multiple regression analysis, SCF was a significant determinant of CD34+ cell levels in BC patients (R = 0.50, p = 0.03) and of VCAM-1 levels in MM patients (R = 0.32, p = 0.02). SCF was negatively correlated with CD34+ cell count in patients with BC. SCF and VCAM-1 blood levels were correlated in MM and BC patients. CONCLUSION: SCF and VCAM-1 could play a role in PBPC mobilization in patients and could be useful measures by which to study patients undergoing a mobilization regimen.

Effect of autologous platelet concentrate in surgery for idiopathic macular hole: results of a multicenter, double-masked, randomized trial. Platelets in Macular Hole Surgery Group.

OBJECTIVE: To evaluate prospectively the efficacy and safety of autologous platelet concentrate (APC) as an adjuvant in surgery for idiopathic macular hole. DESIGN: Multicenter, double-masked, randomized clinical trial. SETTING: Four university-based ophthalmology clinics. PARTICIPANTS: One hundred ten patients with stage 3 or 4 idiopathic full-thickness macular holes of less than 3 years' duration were randomized (53 eyes to the platelet group and 57 eyes to the control group). INTERVENTIONS: Standardized macular hole surgery versus surgery combined with injection of an APC. In all cases, the procedure consisted of three-port pars plana vitrectomy, posterior hyaloid separation, and nonexpansile fluid-gas exchange. After the fluid-gas exchange, patients were randomized to receive either injection of an APC or no adjunctive treatment. After surgery, patients were positioned face down for 12 days. Platelet counts showed that the concentrates contained a mean of 96.106 platelets (range, 82-102). MAIN OUTCOME MEASURES: Anatomic and functional evaluations were performed at 1, 3, and 6 months after surgery in a double-masked fashion by an independent observer. The main outcome was reapposition of the edge of the macular hole 1 month after surgery. Secondary outcomes were anatomic status at 3 and 6 months, changes in Early Treatment Diabetic Retinopathy Study score, and complications. RESULTS: One month after surgery, the anatomic success rate in the platelet group was 52 of 53 (98%; 95% confidence interval, 0.90-1.00) versus 47 of 57 (82%; 95% confidence interval, 0.70-0.91) in the control group (P = 0.009, Fisher's exact test; relative risk, 0.11; 95% confidence interval, 0.01-0.81). Visual acuity was not significantly different between the two groups at any timepoint. There were no complications specifically attributable to the platelet injection. CONCLUSION: Injection of APC improved significantly the anatomic success rate of surgery for idiopathic macular holes of less than 3 years' duration, but postoperative visual acuity of the platelet group was not statistically different from the control group.

[Kasabach-Merritt syndrome on a congenital tufted angioma]. / Syndrome de Kasabach-Merritt sur angiome en touffes congénital.

INTRODUCTION: Kasabach-Merritt syndrome is a very rare disease of infancy, with profound thrombocytopenia and a mild to severe consumption coagulopathy; this biological phenomenon is difficult to control. CASE REPORT: A 1-month old boy had a congenital plaque-like lesion in the calf. It was a biopsy-proven tufted angioma. Five weeks later, Kasabach-Merritt syndrome developed. After failure of ticlopidine + aspirin, and oral betamethasone treatment, thrombocytopenia was cured with vincristine treatment, then the leg lesion slowly continued to shrink after cessation of the treatment. It had disappeared before the age of 1 year. DISCUSSION: We highlighted two points: 1) Kasabach Merritt does not appear as a complication of a classic hemangioma (infantile, "cellular", "capillary", involuting-type), as it has long been thought. In our experience, it develops on a different endothelial cell proliferation, in this case a congenital tufted angioma, but it can also engraft on a kaposiform hemangioendothelioma. 2) These patients are difficult to treat because, up to now, no single treatment has given constant by good results. Vincristine was recently introduced in the treatment of Kasabach-Merritt syndrome, with excellent, rapid outcome. CONCLUSION: What seems a therapeutic progress in a difficult field needs further control.

[Importance of bFGF ("basic fibroblast growth factor") for diagnosis and treatment of hemangiomas]. / Intérêt du dosage du bFGF ("basic Fibroblast Growth Factor") pour le diagnostic et le traitement des hémangiomes.

OBJECTIVES: Hemangiomas of infancy follow a characteristic three-phases course: proliferation, involution, regressed Proliferative endothelial cells predominate during the proliferative phase. Moreover it has been shown that patients with active angiogenesis have elevated levels of urinary bFGF (basic Fibroblast Growth Factor). PATIENTS AND METHODS: Here we report our preliminary results of urinary bFGF assay (ELISA) for the diagnosis and follow up of severe hemangioma. We also assayed bFGF in normal infants, in patients with large vascular malformations and in infants with Kasabach-Merritt syndrome. RESULTS: In the control group, urinary bFGF was elevated in new borns but nul or very low in infants. Urinary bFGF levels were normal, i.e. very low in 4 patients with a vascular malformation. In infants with a clinically proliferative hemangioma, urinary bFGF was elevated in 8 among the 10 studied. bFGF levels guided treatment in 9 patients. Urinary bFGF was elevated in 4 patients with Kasabach-Merritt syndrome. DISCUSSION: Angiogenesis is regulated by angiogenic and inhibitory factors. The angiogenic factor bFGF is an autocrine growth factor for endothelial cells and hemangioma endothelial cells expressing bFGF in their cytosol during the proliferative phase. As suggested by J. Folkman and his group, assay of urinary bFGF appears useful in differentiating between hemangioma and vascular malformation and for follow up of treated patients.

Studies of portal hemodynamics and hepatic oxygen consumption during acute liver allograft rejection.

Hemodynamics and oxygen variables, plasma cytokines, and histological features of a liver tissue sample obtained by transvenous biopsy were evaluated during 65 episodes of acute rejection. The hepatic venous pressure gradient was significantly higher in patients with acute rejection than in those without (5.1+/-0.3 vs. 3.1+/-0.2 mmHg, P<0.01). The increase in pressure gradient was related to the severity of rejection lesions. Hepatic blood flow was significantly lower in patients with than in those without acute graft rejection (1.28+/-0.11 vs. 1.75+/-0.13 L/min, P<0.05). Plasma interleukin-6 levels were significantly increased in patients with acute rejection and positively correlated with pressure gradient values. In patients with acute rejection, a significant decrease in hepatic venous oxygen content (-16%) was associated with a significant increase in hepatic oxygen consumption (+24%), whereas hepatic oxygen transport did not change significantly. In treated patients with a favorable response, the pressure gradient decreased significantly by 46%, but it remained elevated in patients who later developed chronic graft rejection. In conclusion, this study confirms that acute graft rejection may induce an increase in portal pressure, which is related to the severity of rejection lesions. It also shows that acute rejection decreases hepatic blood flow and increases hepatic oxygen consumption. In addition, it suggests that the hepatic venous pressure gradient might be useful to determine the outcome of rejection.

[Kasabach-Merritt syndrome of the leg associated with osteolysis or Gorham sign]. / Syndrome de Kasabach-Merritt d'un membre associé à une ostéolyse ou signe de Gorham.

INTRODUCTION: Kasabach-Merritt syndrome and Gorham's sign are two uncommon and severe, sometimes life-threatening, complications in infants with vascular lesions. Their association has been described in rare cases. CASE REPORT: An infant with a vast congenital angiomatous lesion including an extensive lymphatic component, developed active regional osteolysis then suddenly suffered disseminated intravascular coagulation of the leg. Medical treatment was unsatisfactory. After unsuccessful use of low molecular weight heparin, pentoxifyllin and alpha interferon, amputation of the leg was required to avoid a fatal outcome. DISCUSSION: Kasabach-Merritt syndrome does not develop on classic immature hemangiomas, despite some contradictory statements in the literature. In our case, a complex tumor developed in association with a lymphatic malformation. The association of Kasabach-Merritt syndrome with osteolysis (Gorham's sign) does not appear to be fortuitous. Therapeutic management of these severe complications is difficult and requires case by case analysis.

Are angiogenic factors, cytokines, and soluble adhesion molecules prognostic factors in patients with renal cell carcinoma?

Angiogenesis has an important role in the progression of solid tumors. Therefore, we measured the blood levels (ELISA) of angiogenic factors [basic fibroblast growth factor (bFGF), hepatocyte growth factor/scatter factor, and vascular endothelial growth factor (VEGF)] and soluble adhesion molecules [E-selectin, intercellular adhesion molecule (ICAM-1), platelet endothelial cell adhesion molecule-1, and vascular cell adhesion molecule-1] in 76 consecutive patients with untreated renal cell carcinoma and 41 healthy controls to evaluate their prognostic value. The serum levels of bFGF, hepatocyte growth factor, and VEGF were significantly higher in patients with renal cancer than they were in healthy subjects. bFGF and VEGF values were significantly higher in patients with disseminated cancer (N+ and/or M+) than they were in those with undisseminated (M-N-) cancer: median = 27 pg/ml, range = 5-118, n = 15 versus median = 8 pg/ml, range = 1-149, n = 61 (P = 10(-4)) for bFGF; and median = 883 pg/ml, range = 200-2317, n = 15 versus median = 278 pg/ml, range = 0-1704, n = 61 (P = 0.006) for VEGF. The blood levels of ICAM-1 and vascular cell adhesion molecule-1 were significantly higher, and the levels of E-selectin and platelet endothelial cell adhesion molecule-1 were significantly lower in patients with renal cancer than they were in controls. Plasma ICAM-1 was higher in metastatic patients (M+) than they were in nonmetastatic (M-) patients: median = 687 ng/ml, range = 294-1091, n = 12 versus median = 408 ng/ml, range = 217-1375, n = 64 (P = 10(-4)). ICAM-1 and bFGF blood values were correlated with the size of the primary tumor. The interleukin 6 and tumor necrosis factor-alpha (TNF-alpha) values of these patients have been previously published and are included in the survival analysis. Univariate analysis showed that bFGF, ICAM-1, interleukin 6, and TNF-alpha, before treatment, were prognostic factors. In multivariate analysis for proportional hazard regression, only TNF-alpha was an independent prognostic indicator, with a normal plasma TNF-alpha being highly predictive for a good prognosis in patients with untreated renal cell carcinoma.

Endothelial cell dysfunction secondary to the adhesion of diabetic erythrocytes. Modulation by iloprost.

We previously showed the correlation between the extent of vascular complications and erythrocyte adherence to endothelium in diabetes mellitus. The accumulation of advanced glycation end products (AGEs) on the erythrocyte surface in diabetes mediates their interaction with endothelial cells through a specific endothelial receptor for AGEs (RAGE). Binding of diabetic erythrocytes to endothelial cells resulted in evidence of oxidant stress responsible for a range of cellular perturbations. In the present study, we have investigated the effect of iloprost, a prostacyclin analog, on several activities modified by diabetic erythrocyte-endothelium interaction: 1) generation of oxidant stress based on production of thiobarbituric acid reactive substances (TBARS: control: 2.37 +/- 0.32 versus iloprost: 1.39 +/- 0.005 mumol/10(11) cells), 2) alteration of the endothelial barrier function as measured by an increase permeability to 125I-albumin (control: 13.31 +/- 0.85 versus iloprost: 9.45 +/- 0.7 10(-7) cm/s) of the endothelial cell monolayer, 3) modification of the endothelial cell function showed by an increase in interleukin-6 release (control: 21.66 +/- 3.11 versus iloprost 15.45 +/- 0.76 ng/10(6) cells). The increase in permeability to albumin as well ass TBARS production and interleukin-6 release were inhibited by iloprost (10(-8)-10(-6) mol/l) treatment in a dose-dependent fashion. These results indicate that erythrocyte associated AGEs might alter endothelial cell function. The perturbations can be limited in vitro by iloprost.

Monocyte modulation of endothelial leukocyte adhesion molecules.

Leukocyte adhesion to endothelium is dependent on expression of specialized molecules. Several of these molecules are upregulated by cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). We investigated the effect of medium conditioned by unstimulated (MCM) or stimulated monocytes and of recombinant cytokines on endothelial adhesion receptor expression. IL-1 beta, TNF-alpha, and MCM induced E-selectin similarly, whereas MCM induced VCAM-1 and ICAM-1 to a lesser extent than did TNF-alpha, and MCM induced VCAM-1 only weakly. The addition of pentoxifylline (10(-3) mol/L) to monocytes during MCM preparation blocked TNF-alpha production but not that of IL-1 beta or IL-6, and it reduced IL-1ra significantly (p < 0.05). When the MCM was devoid of TNF-alpha or when TNF-alpha was neutralized with a specific antibody, the action of MCM on E-selectin expression was significantly lower. Anti-IL-1 beta decreased the activity of MCM on endothelial E-selectin expression by about 50%. The effect of MCM on adhesion molecules was accompanied by an increase in monocyte adhesion. Inhibition of TNF-alpha production reduced monocytes adhesion slightly but significantly (18%, p < 0.05), whereas anti-IL-1 beta antibody decreased adhesion by 48% (p < 0.001). These results show that adherent monocytes released cytokines and antagonists that affect leukocyte adhesion receptors on endothelium differently from recombinant cytokines. E-selectin expression--and to a lesser extent ICAM expression--is modified, resulting in a modulation of leukocyte adhesion to endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)