MnTMPyP, a metalloporphyrin-based superoxide dismutase/catalase mimetic, protects INS-1 cells and human pancreatic islets from an in vitro oxidative challenge.

AIMS: Pancreatic islets can be lost early following allotransplantation from oxidative stress. Antioxidant enzyme overexpression could confer a beneficial effect on islets exposed to reactive oxygen species (ROS) and nitrogen species. Here, we tested the effect of MnTMPyP, a superoxide dismutase/catalase mimetic. METHODS: INS-1 insulin-secreting cells or human islets were cultured with MnTMPyP and exposed to a superoxide donor (the hypoxanthine/xanthine oxidase (HX/XO) system), a nitric oxide donor [3-morpholinosydnonimine (SIN-1)] or menadione. Viability of INS-1 cells was assessed by WST-1 colorimetric assay and FACS analysis (Live/Dead test). ROS production was determined using fluorescent probes. Islet viability was estimated by WST-1 assay and endocrine function by static incubation. RESULTS: Following MnTMPyP treatment, ROS production in INS-1 cells was reduced by 4- to 20-fold upon HX/XO challenge and up to 2-fold upon SIN-1 stress. This phenomenon correlated with higher viability measured by WST-1 or Live/Dead test. MnTMPyP preserved islet viability upon exposure to SIN-1 or menadione but not upon an HX/XO challenge. Similarly, decrease in insulin secretion tended to be less pronounced in MnTMPyP-treated islets than in control islet when exposed to SIN-1, but no changes were noticed during an HX/XO stress. CONCLUSIONS: MnTMPyP was able to improve the viability of INS-1 cells and human islets exposed to oxidative challenges in vitro. Protection of INS-1 cells could be as high as 90%. This agent is therefore potentially attractive in situations involving the overproduction of ROS, such as islet transplantation.

Mesenchymal stem cells induce apoptosis of activated T cells.

Mesenchymal stem cells (MSC) have recently been used successfully in humans to control severe graft-versus-host disease. However, the mechanisms involved in their immunomodulatory effects remain a matter of debate. Here, we show that MSC are unable to activate allogeneic T cells even in the presence of T-cell growth factors. We then found that MSC inhibit T-cell proliferation triggered either by allogeneic, mitogenic or antigen-specific stimuli. Interestingly, MSC inhibit T-cell proliferation by inducing apoptosis of activated T cells, but have no effect on resting T cells. Furthermore, we show that this apoptosis could be related to the conversion of tryptophan into kynurenine by indoleamine 2,3-dioxygenase expressed by MSC in the presence of IFNgamma. Moreover, we show that the inhibitory effect of MSC is neither abrogated nor modified during expansion in culture or after irradiation. Together, these results bring new insight to the mechanisms of immunosuppression induced by MSC and might help to develop their clinical use controlling immune-related adverse effects in humans.

Distinct reactivity of Burkitt's lymphoma cell lines with eight monoclonal antibodies correlated with the ethnic origin.

Twenty-eight Burkitt's lymphoma(s) (BL) cell lines were analyzed with anti-human immunoglobulins and monoclonal antibodies: Y29/55, B1, and BA1 are slightly different pan-B-reagents; TU1 and BL13 are two discriminating markers of the follicle; RFT1 is a pan-T-reagent expressed on the follicle mantle; AL2 reacts with the common acute lymphoblastic leukemia antigen gp100; and 38:13 recognizes a BL-associated antigen. Those lines were classified into 3 groups according to their membrane phenotype. In the first 2 groups, cell lines were derived from BL of germinal center origin, whereas in the last group they were established from BL cells originating in the bone marrow. All cell lines in the last group were from Caucasian BL, whereas lines from African BL of a high-incidence area were in group 1. North African cases were in group 2. Those distinct subgroups were not related specifically to the reactivity with Epstein-Barr virus nuclear antigen, the type of chromosomal translocation, or the clinical features. The variations induced by growth culture as well as the clinical implications were discussed.

[Diagnosis of lung cancer. DNA microarrays in thoracic oncology]. / Diagnostic des CBP. Les "puces à ADN" en oncologie thoracique.

DNA microarrays allow simultaneous measurement of the expression of several thousand genes in a biological specimen. This technique represents a major advance in the analysis of tumour biopsies. It may be used to refine the anatomical- pathological diagnosis at a molecular level and thus lead to better diagnostic and prognostic classification and improved therapeutic decisions.

Tumor necrosis factor as an autocrine growth factor for neuroblastoma.

Recombinant tumor necrosis factor (TNF) stimulates the proliferation of two neuroblastoma cell lines, SKNFI and SKNBE, in both serum-free medium and fetal calf serum-supplemented medium but has no effect in medium without insulin. This effect is very similar with TNF doses ranging from 5 to 500 ng/ml but depends on the duration of treatment; when cells are treated for 168 h with TNF, the maximal index of proliferation is observed between 120 and 144 h of treatment. The two neuroblastoma cell lines express type A and type B TNF receptors and contain TNF protein; however, TNF is undetectable in culture supernatants. Treatment of the two neuroblastoma cell lines with a rabbit polyclonal antibody to TNF for 96 h fully inhibits [3H]thymidine incorporation; less than 5% viable cells are left in the samples after treatment. A combination of two monoclonal antibodies against type A and type B TNF receptors also inhibits over 85% of the [3H]thymidine incorporation by the two cell lines after 96 h of treatment; the use of a single antibody has a partial effect, suggesting that both receptors are functional on the neuroblastoma cell lines. Taken together, these results show that TNF is an autocrine growth factor for the two neuroblastoma cell lines SKNFI and SKNBE. The results described above have been confirmed on two other neuroblastoma cell lines, IRM32 and CLB-PE.

Interleukin (IL)-10 and IL-6 are produced in vivo by non-Hodgkin's lymphoma cells and act as cooperative growth factors.

The in vivo production of interleukin (IL)-10, IL-6, IL-2, and tumor necrosis factor (TNF)-alpha in tumor samples was investigated by immunohistochemistry in 54 non-Hodgkin's lymphomas (NHLs). Respectively, 55, 89, 23, and 29% of tumor samples were found positive for IL-10, IL-6, IL-2, and TNF-alpha expression by immunohistochemistry. Using reverse transcription-PCR, the mRNA of IL-10 and IL-6 were detectable in all samples tested and in 90 and 34% of the samples for TNF-alpha and IL-2, respectively. In 13 patients, fresh tumor tissue was available for B NHL cell purification with Dynabeads. IL-10, IL-6, IL-2, and TNF-alpha were detectable in the supernatant of 38, 100, 0, and 23% of purified tumor cell preparations (PTCPs), respectively. All patients with detectable IL-10 in culture had increased serum IL-10. IL-6 production by tumor cells and serum IL-6 levels were also found to be highly correlated (P < 0.0001). This suggests that tumor cells are a major source of serum IL-1O and IL-6 in these patients. Exogenous IL-10, IL-6, IL-2, and TNF-alpha significantly enhanced the [3H]thymidine uptake in 13 of 13 (100%), 5 of 13 (38%), 9 of 13 (69%), and 2 of 10 (20%) PTCPs costimulated with anti-CD40, respectively. IL-2, IL-6, and TNF-alpha synergized with IL-10 in 54, 23, and 30% of PTCPs. The combination of IL-10, IL-2, and IL-6 induced the maximal level of proliferation in 12 (92%) of 13 PTCPs. CD40 ligand mRNA expression was also detectable in vivo using reverse transcription-PCR in 28 of the 29 (97%) tumor samples tested, including 11 of those tested for [3H]thymidine incorporation. These results show that IL-1O, IL-6, IL-2, and TNF-alpha are produced in NHL tumors and may cooperate in vivo to increase NHL cell proliferation.

Correlation between clinical response to interleukin 2 therapy and sustained production of tumor necrosis factor.

Twenty-five previously untreated patients with metastatic renal cell carcinoma were treated with 5-day cycles of continuous infusion of interleukin 2 (IL2) and lymphokine-activated killer cell reinfusion. Five achieved a partial response. Three patients were found to have detectable tumor necrosis factor (TNF) in serum before initiation of therapy. On the fifth day of therapy, 24 patients had circulating TNF with immunoradiometric assay whereas 13 had detectable biological activity. Two days after the end of IL2 therapy, TNF concentration (immunoradiometric assay) decreased in most cases but was still detectable in 17 patients. Thirteen patients had still circulating TNF bioactivity. Although there was no significant difference between TNF levels observed on the fifth day of therapy in the responder and nonresponder groups, 48 h after the end of IL2 infusion, both the TNF concentration and the biological activity were significantly higher in the group of responder patients. This result suggests that the clinical response to IL2 therapy in patients with metastatic renal cell carcinoma is correlated to a sustained production of TNF after the end of IL2 infusion.

Clinical relevance of CD44 cell-surface expression and N-myc gene amplification in a multicentric analysis of 121 pediatric neuroblastomas.

PURPOSE: In contrast to other human tumors, a repression of the cell-surface glycoprotein CD44 on neuroblastoma is a marker of aggressiveness that usually correlates to N-myc amplification. We thus compared the prognostic value of both markers in the initial staging of 121 children treated for neuroblastoma in collaborative institutions. METHODS: Frozen samples were analyzed by a rapid and well-standardized technique of immunostaining with monoclonal antibodies (MoAbs) against epitopes in the CD44 constant region. RESULTS: In this retrospective series, CD44 was expressed on 102 specimens and strongly correlated with favorable tumor stages and histology, younger age, and normal N-myc copy numbers. In univariate analysis, CD44 expression and normal N-myc were the most powerful markers of favorable clinical outcome (P < 10(-6) and chi 2 = 65.40 and P < 10(-6) and chi 2 = 42.56, respectively), but analysis of CD44 affords significant prognostic discrimination in subgroups of patients with or without N-myc-amplified tumors. In the subgroup of stage IV neuroblastomas, CD44 was the only significant prognostic marker (P < .02, chi 2 = 5.76), whereas N-myc status was not discriminant. In multivariate analysis of five factors, ie, N-myc amplification, CD44 expression, age, tumor stage, and histology, the only independent prognostic factors of event-free survival were CD44 expression and tumor stage. CONCLUSION: The analysis of CD44 cell-surface expression must be recommended as an additional biologic marker in the initial staging of the disease.

Promoter methylation of genes in bronchial lavages: a marker for early diagnosis of primary and relapsing non-small cell lung cancer?

A prospective screening program, including CT, autofluorescent bronchoscopy, biopsies and bronchial lavage (BL) collection, was initiated with the specific goal of identifying biomarkers for the early detection of non-small cell lung cancer. We report and discuss the results of p16, DAPK, MGMT, FHIT and APC methylation analysis in the 126 first patients: 77 at high risk of cancer and 49 followed up after primary cancer resection. Positive results were found in 49% of BLs, 53% in current smokers and 43% in former smokers. In presence of peripheral tumours, only 38% of BLs were abnormal versus 73% in presence of central tumours, 50% in presence of preneoplasic lesions and 47% in absence of lesions. FHIT methylation was an early event, observed in one-third of the BLs from patients with or without lesions as well as in tumours. APC methylation was a late event observed in 33% of tumours but rarely in BLs. p16 was methylated in 17% of BLs but in 48% of tumours; DAPK in 15% of BL and 22% of tumours. MGMT methylation was rare. Among patients followed up after cancer surgery, 14 were in remission with normalised BL, whereas three had positive BLs and relapsed with a central tumour. Thus, gene methylation in BL might help to detect central tumours but a CT is crucial for peripheral cancer detection.

Retroviral gene therapy and its application in oncohematology.

A symposium entitled "Foetal and Neonatal Cell Transplantation and Retroviral Gene Therapy" recently organized under the aegis of the Mérieux Foundation in Annecy, France, brought together 100 scientists and clinicians from European countries and the United States. The last day of the meeting focused on retroviral gene therapy in oncohematology. The speakers provided the basis for therapeutic applications of gene transfer and showed practical directions to be followed in the near future. Although it may be mainly restricted to in vitro manipulation of human cells at start, gene therapy is already applicable to the treatment of genetic disorders, cancer, and viral infections. The reality of gene therapy was well illustrated by the nature of the questions raised by clinicians and scientists during the meeting.

Management of pediatric non-Hodgkin's lymphoma.

Over the past 15 years, significant progress has been made in the understanding of the molecular mechanism involved in malignant transformation of lymphocytes as well as in the management and treatment of non-Hodgkin's lymphoma (NHL) in childhood. Cyto-histological classifications and immunophenotyping of different types of NHL have contributed to the characterisation of three major subtypes of NHL in children i.e. Burkitt's lymphoma (BL), lymphoblastic lymphoma (LL) and large cell lymphoma (LCL). Precise staging of the disease at diagnosis is necessary before the onset of the treatment and should be performed as quickly as possible. Presence of bone marrow and central nervous system (CNS) involvement are major prognosis criteria. In most cases, surgery has no therapeutic role and is required only for diagnostic procedures. Similarly, several studies have demonstrated that irradiation of various sites including the CNS does not improve survival. Thus, NHL patients are usually treated with chemotherapy alone. BL and LL have distinct clinical presentations and require completely different chemotherapy protocols. After comparable induction phases with intensive chemotherapy regimens, the former is usually treated with a short consolidation phase while the latter receives a long lasting consolidation consisting of intermittent chemotherapy for at least one year. The prognosis of stage I-II, and III-IV bone marrow negative NHL of children is excellent with respectively 95% and 75% long term survival. However, patients with concomittent CNS and bone marrow involvement in both histological subtypes have a considerably worse prognosis with only 30% long term survival.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of CD44 prognostic value in neuroblastoma: comparison with the other prognostic factors.

CD44 gene products are potential markers of aggressiveness in different tumour models, a result which prompted us to study clinical neuroblastoma (NB) specimens. CD44 expression was determined by immunostaining of 52 tumour samples from newly diagnosed NB with a monoclonal antibody (J173) directed against an epitope common to all CD44 isoforms. CD44 immunoreactivity was detected in 37 of the tumours (71%). CD44 was expressed in all 22 NBs with favourable prognoses (stages 1, 2 or 4S), but only 50% (15/30) of advanced NB (stages 3 and 4) (P < 10(-4)), suggesting that the absence, rather than the overexpression, of CD44 is a signal of tumour aggressiveness. The cumulative progression-free survival was significantly longer in patients with CD44 positive tumours compared with patients with CD44 negative tumours (P < 10(-5)). More importantly, progression-free survival was also significantly higher in CD44 positive patients within the high-risk group (P < 0.01). In univariate analysis, we tested the prognostic value of tumour expression of CD44 in comparison with tumour stage, age, tumour histology, and presence or absence of amplification of the MYCN protooncogene. All five measures had significant prognostic value. The expression of CD44 and the absence of MYCN amplification were the most powerful predictors of a favourable outcome. In a multivariate analysis of these measures, CD44 expression and tumour stage were the only independent prognostic factors for the prediction of patient survival. NB is the first clinical model described in which tumour aggressiveness correlates with repression rather than stimulation of CD44 expression. We recommend the use of CD44 as an additional biological marker in the initial staging of NB.

Clinical relevance of CD44 cell surface expression and MYCN gene amplification in neuroblastoma.

This multicentric analysis of tumours obtained from 140 patients with neuroblastoma confirms that the lack of CD44 expression is a highly significant factor of poor prognosis and, as previously published in multivariate analysis of the four factors, i.e. MYCN amplification, CD44 expression, age and tumour stage, CD44 expression and tumour stage were the only independent prognostic factors of event-free survival (Combaret et al., J Clin Oncol 1996, 14, 25-34). Furthermore, CD44 analysis affords significant prognostic discrimination in subgroups of patients with or without MYCN amplified tumours, both in low-stage neuroblastomas and high-grade neuroblastomas. In the subgroup of patients with low-stage neuroblastoma and the stage 4 subgroup, CD44 was the only independent prognostic factor for the prediction of event-free survival in a multivariate analysis. In conclusion, CD44 is one of the most powerful factors for predicting clinical outcome in neuroblastoma at the time of initial staging.

Use of a liquid cell culture assay to quantify the elimination of Burkitt lymphoma cells from the bone marrow.

A new liquid cell culture assay for propagating Burkitt lymphoma (BL) cells in the presence of excess allogeneic irradiated bone marrow (BM) has been standardized. The Burkitt cell lines used in this assay were newly established from the tumours of our patients and were either EBV(+) or EBV(-). The phenotypic characteristics of lines were similar to those of the original malignant tumours. With the help of this liquid assay it was possible to evaluate the efficacy of in vitro procedures designed to completely eliminate malignant cells from harvested BM prior to autologous transplantation (i.e., immunomagnetic depletion or antibody-complement-mediated cytolysis). With six out of the seven cell lines tested, the assay permitted the detection of as few as one BL cell in 4 X 10(6) normal BM cells and the measurement of up to 5 log BL cell elimination from 1% contaminated samples.

Detection of minimal malignant cell infiltration in the bone marrow of patients with solid tumours, non-Hodgkin lymphomas and leukaemias.

Detection of a minimal malignant cell infiltration is becoming essential in oncohaematology, either as a prognostic factor in solid tumours or as a criterion of response to therapy in both haematological and non-haematological malignant proliferations. Evaluation of the efficiency of new therapeutic protocols, especially those involving high-dose chemotherapy and allogeneic or autologous bone marrow transplantation, requires agreement between the different investigators on the definition of minimal residual disease in the bone marrow, on the limits of detection of the various available methods and on the types of investigations required in each clinical situation. This report summarizes the conclusions reached at the First France-Autogreffe Meeting on Minimal Residual Disease in the Bone Marrow.

S-L 11.14: a monoclonal antibody recognizing neuroectodermal tumors with possible value for bone marrow purging before autograft.

A new monoclonal antibody (S-L 11.14) raised against small cell lung cancer reacted with all but one neuroblastoma tumor cell sample tested and was relatively specific for such cells within the bone marrow. The ability of S-L 11.14 to eliminate neuroblastoma cells from the bone marrow with an immunomagnetic purging method was evaluated in an experimental model using the LAN.1 and SKNBE neuroblastoma cell lines as targets. Residual malignant cells after the purging procedure were quantified by the Hoechst staining method. The use of S-L 11.14 as a single reagent resulted in a 3-log elimination of malignant cells, a depletion equal to that obtained with a cocktail of five monoclonal antibodies currently used in clinical trials. The addition of the S-L 11.14 antibody to this cocktail did not enhance depletion.

Systemic interleukin-2 therapy in children with progressive neuroblastoma after high dose chemotherapy and bone marrow transplantation.

Two children with active metastatic neuroblastoma after high dose chemotherapy and bone marrow transplantation (BMT) received a high dose continuous infusion of interleukin-2 (IL2) 120 days after an autologous BMT for patient 1 and 90 days after an allogeneic non T cell-depleted BMT for patient 2. Usual side effects of IL2 therapy were observed without life-threatening complications or any major hematological toxicity. The reactivation of graft-versus-host disease during IL2 infusion in patient 2 was the major BM-related complication but it improved with IL2 interruption and corticosteroids. IL2 induced a complete remission (9+ months) in patient 1 with the disappearance of bone metastases and local tumor but patient 2 progressed after cessation of therapy. Patient 1 presented with a large excess of circulating NK cells in the period after autologous BMT and IL2 induced a preferential outgrowth of this lymphocyte subset.

Protection of insulin-secreting INS-1 cells against oxidative stress through adenoviral-mediated glutathione peroxidase overexpression.

OBJECTIVES: A large fraction of an islet graft can be lost early following allotransplantation from various non specific mechanisms including oxidative stress. Overexpression of antioxidant enzymes could confer a beneficial effect on islets exposed to reactive oxygen and nitrogen species. We examined the viability of beta cells driven to overexpress glutathione peroxidase (GPx) and exposed to a superoxide donor (hypoxanthine/xanthine oxidase HX/XO) and a nitric oxide donor (3-morpholinosydnonimine SIN-1). METHODS: Cultured INS-1 rat-derived insulin-secreting cells were transfected by an E1-deleted adenovirus carrying GPx cDNA (AdGPx). Additional experiments were performed with an adenovector carrying Cu/Zn superoxide dismutase cDNA (AdSOD). Cellular viability was tested by the WST-1 colorimetric assay and functionality by static incubation. RESULTS: AdGPx increased GPx activity within 48 hours from 0 (untransfected cells) to 60 +/- 11 U/g (cells transfected at an MOI of 25: 1). GPx overexpression significantly reduced cytotoxicity induced by HX/XO from 10.81 +/- 1.41 to 5.42 +/- 2.62% at 10 mU/ml and from 61.19 +/- 4.17 to 52.9 +/- 4.39% at 20 mU/ml (p=0.0002, transfected cells vs control cells). Doses of SIN-1 from 600 to 1000 micromol/l resulted in cytotoxicity ranging from 17.66 +/- 3.48 to 45.97 +/- 6.48% in control cells and from 5.65 +/- 1.37 to 35.80 +/- 5.59% in AdGPx transfected cells (p=0.015). The combination of AdGPx and AdSOD did not exhibit any synergistic cytoprotective effect. Control cells exposed to a HX/XO stress exhibited a reduction in glucose-theophylline stimulated insulin secretion by half, while stressed GPx overexpressing-cells maintained the same insulin secretion level than non-stressed cells. CONCLUSIONS: Adenoviral-induced overexpression of GPx enhances the resistance of a rat beta cell line to both reactive oxygen (ROS) and reactive nitrogen species (RNS) cytotoxicity. Transposition of these findings to human islet transplantation with a clinically-relevant procedure deserves further investigations.

Study of blood lipids in 30 children with a malignant hematological disease or carcinoma.

Levels of total lipids, cholesterol and triglycerides were followed in 30 children with malignant hematological diseases, or solid tumors; in addition, chylomicrons, VLDL, LDL and HDL were separated by electrophoresis on polyacrylamide gel. Nine of the 13 children with acute lymphoblastic leukaemia presented an increase of triglycerides and VLDL at time of diagnosis; all other parameters (cholesterol, HDL, LDL, chylomicrons) were normal. The lipid profile went back quickly to normal when hematological remission was obtained and remained stable throughout the course of the disease whatever the evolution might be. Five more patients in relapse were studied and did not present abnormalities. Similar results were obtained in 1/2 patients with acute myeloblastic leukaemia and in 3/5 patients with lymphoma. No lipid profile abnormality was observed in four patients with solid tumor and in one patient with histiocytosis X. The values of triglycerides and VLDL in pathological cases will be given. The endogenous nature of this disturbance is discussed as well as its relation with the rate of malignant lymphoid proliferation.

[Biological markers for the prognosis of neuroblastoma: proposal of a method of analysis]. / Marqueurs biologiques pour le pronostic du neuroblastome: proposition d'une méthodologie d'analyse.

The prognosis of pediatric neuroblastoma depends both on clinical presentation and on certain cellular and molecular characteristics. At the present time, two hypotheses can be drawn to explain both clinical and biological heterogeneity. In the first hypothesis, neuroblastoma progresses from early to late clinical stages through a classical multistep process linked to an accumulation of molecular abnormalities. In the second hypothesis, neuroblastoma represents an heterogeneous group of unrelated diseases, where most of stages I and II or stage IVS neuroblastomas can rather be considered as benign tumors, and stage IV neuroblastoma as a true malignant proliferation. To ascertain relevant biological factors for the prognosis of the disease, it is uppermost important that all investigators agree on biological criteria for analysis when neuroblastoma tissue is available in screened and unscreened populations. This paper reviews the biological tools available for prognosis in neuroblastoma, the priority for analysis of biological markers according to reliability, feasibility, and reproducibility of analysis procedure, and the conditions of tissue storage for further analysis of these biological markers. The standardized biological evaluation of neuroblastoma will allow, first, to collect sufficient data for multivariate analysis of prognostic factors and, second, to better define the putative links between various forms of the disease.