Proteomic profile of pre - B2 lymphoblasts from children with acute lymphoblastic leukemia (ALL) in relation with the translocation (12; 21).

BACKGROUND: Until now, the major prognostic factors for pediatric acute lymphoblastic leukemia (ALL), age, white blood cell count and chromosomal alterations are initially taken into account for the risk stratification of patients. In the light of protein marker studies to classify subtypes of Acute Myeloblastic Leukemia efficiently, we have compared the lymphoblastes proteome in Childhood ALL in accordance with the presence of t(12;21), indicator of good prognosis, usually. METHODS: Protein expression in pre-B2 lymphoblastic cells, collected from residual bone marrow cells after diagnostic procedures, was analyzed using two dimensional gel electrophoresis protocol. Protein spots whose average normalized volumes were statistically different in the two patients groups (n = 13; student t test p < 0.01), were excised. Tryptic peptides were then analyzed using a nano-LC1200 system coupled to a 6340 Ion Trap mass spectrometer equipped with a HPLC-chip cube interface. The tandem mass spectrometry peak lists extracted using the DataAnalysis program, were compared with the protein database Mascot Daemon. RESULTS: We focused on twelve spots corresponding to sixteen identified candidate proteins among the 26 found differentially expressed (p ≤ 0.05) regarding the presence of t(12;21). Among over expressed proteins, two proteins were implicated in cellular growth arrest (i.e. calponine 2, p ≤ 0.001 and phosphatidylinositol transfer protein beta, p ≤ 0.001) in accordance with good prognosis, while two other proteins favored cell cycle proliferation (i.e. methionine adenosyl transferase 2ß, p ≤ 0.005 and heterogeneous nuclear ribonucleo-proteins A2 p ≤ 0.01) and could therefore be good marker candidates of aggressiveness. Level of expression of proteasome subunit beta type-2 (p ≤ 0.01) and protein casein kinase 2α (p ≤ 0.01) which both favored apoptosis, deubiquitinating enzyme OTUB1 (p ≤ 0.05) and MLL septin-like fusion protein MSF-B, septin 9 i4 (p ≤ 0.01) were in accord with a good prognosis related to t(12;21) lymphoblasts. CONCLUSION: By drawing up the protein map of leukemic cells, these new data identified marker candidates of leukemic aggressiveness and new t(12;21) patients subgroups. These preliminary results will be in the near future confirmed by using a larger sample of pre-B2 childhood ALLs from national lymphoblastic cell collections.

Importance of local hypoxia on endothelial phenotype for an in vitro approach to bone marrow angiogenesis.

The vasculature of bone marrow differs from that in other organs, and its characteristics should be considered when exploring the medullar angiogenesis associated with hematological malignancies. We show here that the human bone marrow sinusoidal cell line HBME-1 has a specific expression pattern of angiogenic factors and receptors, characterized by a unique VEGFR3(+), Tie2(-) signature, that resembles the in vivo pattern. Moreover, the HBME-1 cultured for up to 3 days in hypoxic conditions, similar to those found in the bone marrow, specifically downregulated expression of VEGFR1, VEGFR2 and ETAR. Thus, a model using bone marrow sinusoidal cells cultured under reduced oxygen tension may be more relevant than classical in vitro endothelial cultures for understanding the interactions between endothelial and malignant cells in the medullar microenvironment.

Rac-1 GTPase controls the capacity of human leukaemic lymphoblasts to migrate on fibronectin in response to SDF-1α (CXCL12).

Acute lymphoblastic leukaemia (ALL) is characterized by malignant cell infiltration of bone marrow, requiring chemotactic response to SDF-1α. Using time-lapse video, we measured the velocity of ALL cells on fibronectin, and found that SDF-1α increased their migration activity for 2 h, but had no effect after 4h, following internalization of its receptor CXCR4. Transfection of ALL cells with dominant-negative Rac1 mutant significantly prolonged their chemotactic response to SDF-1α, and this effect was associated with an alteration of CXCR4 internalization. These data suggest a regulatory role for Rac1 in the chemotactic response of ALL cells to SDF-1α via receptor processing.

In vitro secretion of matrix metalloprotease 9 is a prognostic marker in childhood acute lymphoblastic leukemia.

Matrix metalloproteases (MMPs) are endopeptidases involved in tumor cell invasion. Childhood acute lymphoblastic leukemia (ALL) is characterized by its capacity to infiltrate different organs. We analyzed the expression of MMP-2, -9, -14 and TIMP-1 and -2 in a prospective study on 86 children with newly diagnosed ALL (73 B- and 13 T-lineage) and 9 children at relapse with B-ALL. Membrane-bound and intracytoplasmic MMPs and TIMPs were analyzed by flow cytometry, and secreted MMPs were quantified by ELISA. In patients at relapse, MMP-14 was present in a greater proportion of the B-ALL cell population than at diagnosis. In patients with peripheral infiltration, intracytoplasmic MMP-9 was significantly higher than in patients without infiltration. ROC curve and Kaplan-Meier curve analysis showed that a high secretion of MMP-9 (>2450 pg/ml/10(6) cells) was associated with a lower overall survival rate, suggesting that the secretion of MMP-9 is an independent prognostic factor in childhood B-ALL.

Increased levels of tissue factor activity and procoagulant phospholipids during treatment of children with acute lymphoblastic leukaemia.

The use of L-asparaginase (L-ASP) in paediatric patients with acute lymphoblastic leukaemia (ALL) is associated with thrombotic complications. We evaluated the activities of tissue factor (TFa), thrombomodulin (TMa) and procoagulant phospholipids (PPL) in 26 consecutive children with ALL (25 B-ALL and one T-ALL) treated by the French Acute Lymphoblastic Leukemia group (FRALLE)-2000 protocol. Samples were obtained at diagnosis, after glucocorticoid (GC) therapy, during the induction phase with L-ASP, vincristine (VCR) and adriamycin (ADR), during the re-induction and within the week after treatment. Plasma levels of TFa, TMa and PPL increased gradually and significantly during the different phases of the treatment, with higher levels observed during the induction period, and decreased after treatment discontinuation. In vitro studies showed that the different drugs used for ALL treatment could induce a weak expression of TF and procoagulant activity (PCA) on normal and leukaemia blood cells, while a marked effect was observed on endothelial cells. In conclusion, these data indicate that, in addition to the well-identified increased in coagulation factors and inhibitor deficiencies, the injury of the endothelium could lead to the release of TF and PPL and could contribute to the hypercoagulability of children treated for ALL.

Plasma and liver hepatitis C virus variability in patients coinfected with human immunodeficiency virus.

Liver and plasma hepatitis C virus (HCV) variability was compared by E2 cloning and sequencing in three patients coinfected with HCV and human immunodeficiency virus (HIV) before and after interferon treatment and in three patients solely infected with HCV. The plasma and liver samples contained unique sequences. In the patients coinfected with HIV, accumulated random mutations produced mostly nonsynonymous substitutions in contrast to the reduced HCV genetic variability seen after treatment.

Human endothelial cells synthesize protein Z, but not the protein Z dependent inhibitor.

Protein Z (PZ) is a vitamin K-dependent protein isolated from human plasma, and acts as a cofactor for a serpin, called protein Z-dependent protease inhibitor (ZPI). A prothrombotic phenotype has been reported in PZ deficient mice, and PZ deficiencies have been observed in patients with arterial thrombotic events. PZ was immunologically detected in the endothelium of atherosclerotic arteries, suggesting that endothelial cells could be involved in the production of PZ. In this study we analyzed the synthesis and release of PZ and ZPI by human umbilical vein endothelial cells (HUVEC), representative of the macrovasculature, and by HMEC-1, a microvascular endothelial cell line. PZ was quantified by a specific ELISA in the supernatant and in the lysates of both cellular types. Western blotting of the supernatants showed the presence of a band of 62 kDa, identical to PZ synthesized by the hepatoma cell line HepG2. mRNA of PZ was also detected in each cellular type. PZ biosynthesis was unaffected by inflammatory cytokines in HUVEC, whereas a slight decrease of mRNA and PZ antigen (53.5 +/- 14.5% of protein synthesis as compared to the control, p < 0.01) and a modest increase (126 +/- 8.5% as compared to the control, p < 0.05) were induced respectively byTumor Necrosis Factor (TNF)-alpha (25 ng/ml) and oncostatin M (5 ng/ml) in HMEC-1. Immunological studies showed the presence of PZ near the nucleus and a possible expression of PZ at the membrane. In addition, PZ was present in the endothelial cells of both normal arterial and venous vessel sections. In contrast, neither ZPI nor its mRNA was detected in endothelial cells.

Spontaneous and cytokine-evoked production of matrix metalloproteinases by bone marrow and peripheral blood pre-B cells in childhood acute lymphoblastic leukaemia.

The present work focused on the study of the secretory activity of pre-B acute lymphoblastic leukaemia (ALL) cells harvested from bone marrow (BM) and peripheral blood (PB) in 16 children. The basal and cytokine (SDF-1, GM-CSF, bFGF, VEGF)-stimulated secretions of gelatinases 2 and 9 (MMPs-2 and -9) and expression of their genes were monitored by zymography and RT-PCR, respectively. A wide heterogeneity was found in the secretory capacities of these cells. The basal secretion of MMP-9 was more frequently observed than that of MMP-2 in both cell types. The cytokines VEGF and bFGF were found to induce predominant stimulatory effects on the MMP-2 secretion. In contrast, GM-CSF was shown to exert a more pronounced activation of the MMP-9 production. Experiments using inhibitors of metabolic pathways (U0126, LY294002 and SN50) revealed that the secretion of MMP-9 was mediated through PI3/MEK1 kinases. The MMP-2 secretion appeared to be however, stimulated through a different metabolic pathway. The microfluorimetric approach showed that the basal and stimulated secretions of MMPs-2 and -9 depended on the extracellular calcium pool. The cytokines VEGF and bFGF represent potent factors increasing the intracellular calcium concentration with similar kinetics. In contrast, GM-CSF was found to activate a verapamil-sensitive efflux of indo-1 from cytosol suggesting that this cytokine could be responsible for the activation of xenobiotic membrane transporters. Experiments using the trypan blue exclusion test demonstrated that bFGF, in contrast to VEGF and GM-CSF, markedly augmented pre-B ALL cell survival. Further investigations into a possible correlation between the plasma concentrations of MMP-2 and -9, VEGF, bFGF and GM-CSF, and the poor evolution of pre-B ALL in children could have valuable diagnostic implications.

Sequences of clustered epitopes in Gag and Nef potentially presented by predominant class I human leukocyte antigen (HLA) alleles A and B expressed by human immunodeficiency virus type 1 (HIV-1)-infected patients in Vietnam.

The aim of this study was to define pluriepitopic regions in Gag and Nef possibly relevant in the perspective of a vaccine design in a vietnamese population. The protein sequences derived from gag and nef genes and phenotyping of the class I human leukocyte antigens (HLA) A and B alleles were established for 28 human immunodeficiency virus type 1 (HIV-1)-infected patients from Ho Chi Minh City, Vietnam. The protein sequences display polymorphism mutations as compared with a B reference strain (HXB2). The most frequently represented HLA-A and -B alleles were HLA-A11, A02, and A33 expressed by 35.7, 23.2, and 21.4% of the patients, respectively, and HLA-B75, B46, and B62 expressed by 35.7, 25, and 17.9% of the patients, respectively. This study allows us to determine four pluriepitopic regions in Gag and Nef that should be chosen for a vaccine design in a Vietnamese population.

Modifications of T-lymphocyte subsets before and during interferon and ribavirin treatment for chronic hepatitis C infection.

Our purpose was to determine in HCV-infected patients whether T-lymphocyte sub-populations were modified before and during interferon-alpha and ribavirin treatment, and whether this correlated with virological response. Twenty-two naive patients were given IFN-alpha 3 Million Units three times per week for 24 or 48 weeks and ribavirin. Sustained virological response corresponded to undetectable serum HCV RNA at treatment completion and 6 months later. Total blood lymphocyte counts and CD3(+)CD4(+), CD3(+)CD8(+), CD3(+)CD4(+)HLA-DR(+), and CD3(+)CD8(+)HLA-DR(+) lymphocyte subsets evaluated before, during, and after treatment were compared to values from 37 healthy subjects. At inclusion, patients and controls had similar total lymphocyte counts. CD3(+)CD4(+) counts and percentages were significantly higher in HCV patients. HLA-DR expression was also increased in CD4(+) (p < 0.0001) and CD8(+) T-cells (p = 0.0008) as compared with controls. During treatment, all lymphocyte subset counts and percentage decreased except the CD3(+)CD4(+) T-cell percentage which increased. Moreover, after 1 month of treatment, virological responders exhibited higher CD4(+) counts than nonresponders (p = 0.025), whereas they did not differ at inclusion or during the 2nd to 6th months of treatment. After treatment completion, all populations returned to baseline values. These results suggest that CD3(+)CD4(+) T-lymphocyte percentage increase under treatment could be related to IFN immunomodulation and associated with virological response.

Occult hepatitis B virus infection in HIV-infected patients with isolated antibodies to hepatitis B core antigen: Aquitaine cohort, 2002-2003.

We prospectively assessed the prevalence of occult hepatitis B virus (HBV) infection by investigating HBV replication in 160 human immunodeficiency virus (HIV)-infected patients with isolated antibodies to hepatitis B core antigen. This prevalence was 0.6% (1 case/160 patients; 95% confidence interval, 0%-3.4%). A second serum sample was collected later from 52 of the patients. HBV DNA was once again undetectable in all patients, except for the sole patient who had previously been found to be HBV DNA positive.

Hepatitis C virus genetic variability in 52 human immunodeficiency virus-coinfected patients.

The aim of this study was to examine whether hepatitis C virus (HCV) pretreatment quasispecies complexity was linked to virological response or other clinical and biological parameters, in human immunodeficiency virus (HIV)-coinfected patients undergoing anti-HCV treatment. In addition, HCV quasispecies composition is described longitudinally in these patients before, during, and after treatment. The 52 HIV-coinfected patients were included in a randomized therapeutic trial. At inclusion, they had CD4(+) counts of >250/micro l, HIV plasma load of <10,000 copies/ml, and chronic HCV infection with genotype 1 (n = 27), 2 (n = 2) or 3 (n = 23). These values were compared at baseline with 32 HCV-only-infected, interferon-naive patients who were infected with genotype 1, 2, or 3 (n = 16, 1, or 15, respectively). HCV complexity was studied by single-strand conformation polymorphism (SSCP) in E2 hypervariable region 1 (HVR1), and diversity was evaluated at inclusion in 20 coinfected patients by sequencing four major SSCP bands. The baseline number of SSCP bands was identical in HIV-infected and control patients. In HIV-infected patients, HCV complexity was not predictive of sustained virological response to anti-HCV treatment and was unrelated to epidemiological factors, immunological parameters linked to HIV infection (CD4(+) counts, T CD4(+) proliferative responses to HIV-1 p24), protease inhibitor treatment, HCV plasma load, or genotype. HCV diversity was lower in genotype 2- and 3-infected patients. Six months after completion of the anti-HCV treatment, in comparison with baseline, SSCP profiles were modified in 13 of the 21 nonresponding coinfected patients with analyzable samples. In conclusion, in HIV-infected patients, HCV variability had no significant influence on virological response to anti-HCV treatment.

CD4 T lymphocyte proliferative responses to hepatitis C virus (HCV) antigens in patients coinfected with HCV and human immunodeficiency virus who responded to anti-HCV treatment.

CD4 T lymphocyte proliferative responses to hepatitis C virus (HCV) antigens were evaluated before and during an anti-HCV regimen (interferon-alpha2a and ribavirin) in 36 patients coinfected with HCV and human immunodeficiency virus (HIV), to determine whether immune responses against HCV antigens are present in such patients, whether these responses are modified by anti-HCV treatment, and whether they are correlated with treatment efficacy. The CD4 responses against HCV antigens (primarily core antigens) detected at study entry in one-half of the patients did not correlate with anti-HCV treatment efficacy. Of 36 patients, 8 had patterns of persistent immune response to infection by genotypes 3 or 4 that were significantly correlated with sustained virologic response. Persistent immunologic reactivity and sustained virologic response coexisted only in patients infected with genotype 3. These findings suggest that HCV genotype may influence specific immune response, which, in turn, is implicated in virologic control.