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.

Human platelet suspension stimulates porcine retinal glial proliferation and migration in vitro.

PURPOSE: To characterize the cellular and molecular mechanisms underlying the efficacy of autologous platelet suspension adjuvant therapy in the treatment of macular hole. METHODS: Platelet suspensions were: paid from whole blood samples obtained from informed volunteers. For proliferation assays, platelet suspensions or purified growth factors were added to semi-confluent cultures of porcine real glial cells for 24 hours, followed by [3H]thymidine for 15 hours, after which time cells were washed, solubilized, and counted for uptake of radioactive tracer. For cell migration assays, confluent glial cultures were scrape wounded and maintained in the presence or absence of platelet suspension or identified platelet constituents. Cell migration into the denuded area was scored as a function of time. In certain cases, specific pharmacologic inhibitors of growth factor action were added at the same time as platelet adjuvant or growth factors. RESULTS: Platelet suspension adjuvant induced strong mitogenic and chemotactic responses in cultured glia, in a dose-dependent manner. Maximal incorporation of thymidine was two- to threefold that of control levels, with an ED50 approximately 5 x 10(6) platelets/ml, and migration was enhanced up to 80-fold after 48 hours. Platelet suspension-induced proliferation was completely blocked by addition of 25 microM genistein, a tyrosine kinase receptor inhibitor. However, the same concentration only partially blocked the cell migration response. Addition of any single growth factor or protein identified from ELISA analysis, or a combination of all factors, did not significantly stimulate proliferation or cell migration. CONCLUSIONS: Human platelet suspensions exert both proliferative and chemotactic influences on retinal glial cells in vitro, suggesting that the same responses may occur in platelet-induced macular hole repair in humans. Growth factors or proteins that have been identified within the suspensions do not mimic these responses in vitro, implying that additional currently unidentified trophic activities are also present.

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.

Chlamydia trachomatis growth stimulates interleukin 8 production by human monocytic U-937 cells.

Growth of Chlamydia trachomatis serotypes L2 and L3 in a human monocytic cell line, U-937, increased the rate of interleukin 8 (IL-8) release 100-fold. Heat-killed chlamydiae induced a 10-fold-lower level of production of IL-8. IL-8 may play an important role in the inflammatory reaction to chlamydial infection.

Advanced glycation end products, oxidant stress and vascular lesions.

The formation of advanced glycation end products (AGEs) is observed in conditions such as diabetes mellitus and ageing, both associated with vascular disorders. AGEs form by the interaction of an aldose with NH2 of proteins, and the subsequent Amadori rearrangement leads to complex molecules. The heterogeneous class of AGE molecules is found in plasma, cells and tissues and accumulates in the vessel wall and the kidney. AGE reactions can generate reactive oxygen intermediates (ROIs), which can act as signal mediators and can be deleterious for molecules or cells. The AGEs and ROI-induced cellular dysfunctions can interfere with the gene expression of peptides and cytokines regulating cell proliferation and vascular functions. The interaction of AGEs with the AGE receptor (RAGE) is followed by a series of intracellular modifications that may be involved in the development of atherosclerosis. An attempt to minimize AGE formation and to limit ROI production by an appropriate therapy may result in the reduction or slowing of vascular disease in patients with diabetes mellitus.