Effects of fusariotoxin co-exposure on THP-1 human immune cells.

Deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T2), fumonisin B1 (FB1), zearalenone (ZEA), and moniliformin (MON) mycotoxins are common food and feed contaminants produced by Fusarium spp. However, while they are usually found to co-occur in a large range of commodities, only few data are available on mycotoxin co-exposure effects and cellular response mechanisms. In this study, the individual and combined toxic effects of these fusariotoxins were evaluated on the THP-1 human immune cell line as major fusariotoxins are mostly potent immunomodulators. In particular, four relevant fusariotoxin mixtures, namely DON-MON, DON-FB1, DON-ZEA, and NIV-T2, were studied using several parameters including cell viability as well as the expression of cell surface markers and the main mitogen-activated protein kinases (MAPKs). After 48 h exposure, a reduction of cell viability in a dose-dependent manner was observed for T2, the most cytotoxic mycotoxin, followed by NIV, DON, MON, FB1, and ZEA. Regarding mycotoxin mixtures, they mainly showed antagonism on cell viability reduction. Interestingly, at concentrations inhibiting 50% of cell viability, most viable cells exhibited surface marker loss and thus became potentially non-functional. In addition, during the first 18 h of exposure, the effects of mycotoxin mixtures on early cell apoptosis and necrosis were found to be different from those induced by the toxins alone. At the molecular level, after 1 h exposure of individual and combined mycotoxins, the three main MAPK signaling pathways (p38, SAPK/JNK, and ERK1/2) were activated, highlighting a fast reaction of the exposed cells even at low cytotoxicity levels.

Antitumoral effects of cyclin-dependent kinases inhibitors CR8 and MR4 on chronic myeloid leukemia cell lines.

BACKGROUND: Although Imatinib mesylate has revolutionized the treatment of chronic myeloid leukemia, some patients develop resistance with progression of leukemia. Alternative or additional targeting of signalling pathways deregulated in Bcr-Abl-driven chronic myeloid leukemia may provide a feasible option for improving clinical response and overcoming resistance. RESULTS: In this study, we investigate ability of CR8 isomers (R-CR8 and S-CR8) and MR4, three derivatives of the cyclin-dependent kinases (CDKs) inhibitor Roscovitine, to exert anti-leukemic activities against chronic myeloid leukemia in vitro and then, we decipher their mechanisms of action. We show that these CDKs inhibitors are potent inducers of growth arrest and apoptosis of both Imatinib-sensitive and -resistant chronic myeloid leukemia cell lines. CR8 and MR4 induce dose-dependent apoptosis through mitochondrial pathway and further caspases 8/10 and 9 activation via down-regulation of short-lived survival and anti-apoptotic factors Mcl-1, XIAP and survivin which are strongly implicated in survival of Bcr-Abl transformed cells. CONCLUSIONS: These results suggest that CDK inhibitors may constitute a complementary approach to treat chronic myeloid leukemia.

Hepatotoxicity of fusariotoxins, alone and in combination, towards the HepaRG human hepatocyte cell line.

While the reality of mycotoxin co-occurrence in food commodities is now established, their effects in mixtures are not well studied. The present study investigated the individual and combined effects of deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T2), fumonisin B1 (FB1), zearalenone (ZEA) and moniliformin (MON) fusariotoxins on cell viability and cell death mechanisms in proliferating HepaRG cells, a human derived liver cell line. In addition, DON-ZEA being one of the most widespread mycotoxin mixtures in grains worldwide, its effect on the expression levels of genes encoding for sets of hepatocyte-specific functions was studied. After 48 h, T2 appeared to be the most cytotoxic tested fusariotoxins, followed by NIV, DON and ZEA. Furthermore, at low cytotoxic doses, all tested fusariotoxin mixtures (DON-MON, DON-FB1, DON-ZEA and NIV-T2) acted synergistically on cell death. Interestingly, during the first 18 h of exposure, only FB1 and ZEA alone and in combination with DON seemed to induce cell apoptosis and necrosis. At the gene level, after only 1 h, DON-ZEA combination induced expression of drug-metabolizing enzymes contrary to individual exposures. Thus, the observed synergy of fusariotoxin mixtures suggested that their simultaneous presence in food commodities can induce a toxic risk that should be better taken into consideration.

Single Muscle Immobilization Decreases Single-Fibre Myosin Heavy Chain Polymorphism: Possible Involvement of p38 and JNK MAP Kinases.

PURPOSE: Muscle contractile phenotype is affected during immobilization. Myosin heavy chain (MHC) isoforms are the major determinant of the muscle contractile phenotype. We therefore sought to evaluate the effects of muscle immobilization on both the MHC composition at single-fibre level and the mitogen-activated protein kinases (MAPK), a family of intracellular signaling pathways involved in the stress-induced muscle plasticity. METHODS: The distal tendon of female Wistar rat Peroneus Longus (PL) was cut and fixed to the adjacent bone at neutral muscle length. Four weeks after the surgery, immobilized and contralateral PL were dissociated and the isolated fibres were sampled to determine MHC composition. Protein kinase 38 (p38), extracellular signal-regulated kinases (ERK1/2), and c-Jun- NH2-terminal kinase (JNK) phosphorylations were measured in 6- and 15-day immobilized and contralateral PL. RESULTS: MHC distribution in immobilized PL was as follows: I = 0%, IIa = 11.8 ± 2.8%, IIx = 53.0 ± 6.1%, IIb = 35.3 ± 7.3% and I = 6.1 ± 3.9%, IIa = 22.1 ± 3.4%, IIx = 46.6 ± 4.5%, IIb = 25.2 ± 6.6% in contralateral muscle. The MHC composition in immobilized muscle is consistent with a faster contractile phenotype according to the Hill's model of the force-velocity relationship. Immobilized and contralateral muscles displayed a polymorphism index of 31.1% (95% CI 26.1-36.0) and 39.3% (95% CI 37.0-41.5), respectively. Significant increases in p38 and JNK phosphorylation were observed following 6 and 15 days of immobilization. CONCLUSIONS: Single muscle immobilization at neutral length induces a shift of MHC composition toward a faster contractile phenotype and decreases the polymorphic profile of single fibres. Activation of p38 and JNK could be a potential mechanism involved in these contractile phenotype modifications during muscle immobilization.

Recombinant anti-CD4 antibody 13B8.2 blocks membrane-proximal events by excluding the Zap70 molecule and downstream targets SLP-76, PLC gamma 1, and Vav-1 from the CD4-segregated Brij 98 detergent-resistant raft domains.

The biological effects of rIgG(1) 13B8.2, directed against the CDR3-like loop on the D1 domain of CD4, are partly due to signals that prevent NF-kappaB nuclear translocation, but the precise mechanisms of action, particularly at the level of membrane proximal signaling, remain obscure. We support the hypothesis that rIgG(1) 13B8.2 acts by interfering with the spatiotemporal distribution of signaling or receptor molecules inside membrane rafts. Upon cross-linking of Jurkat T lymphocytes, rIgG(1) 13B8.2 was found to induce an accumulation/retention of the CD4 molecule inside polyoxyethylene-20 ether Brij 98 detergent-resistant membranes at 37 degrees C, together with recruitment of TCR, CD3zeta, p56 Lck, Lyn, and Syk p70 kinases, linker for activation of T cells, and Csk-binding protein/phosphoprotein associated with glycosphingolipid adaptor proteins, and protein kinase Ctheta, but excluded Zap70 and its downstream targets Src homology 2-domain-containing leukocyte protein of 76 kDa, phospholipase Cgamma1, and p95(vav). Analysis of key upstream events such as Zap70 phosphorylation showed that modulation of Tyr(292) and Tyr(319) phosphorylation occurred concomitantly with 13B8.2-induced Zap70 exclusion from the membrane rafts. 13B8.2-induced differential raft partitioning was epitope, cholesterol, and actin dependent but did not require Ab hyper-cross-linking. Fluorescence confocal imaging confirmed the spatiotemporal segregation of the CD4 complex inside rafts and concomitant Zap70 exclusion, which occurred within 10-30 s following rIgG(1) 13B8.2 ligation, reached a plateau at 1 min, and persisted until the end of the 1-h experiment. The differential spatiotemporal partitioning between the CD4 receptor and the Zap70-signaling kinase inside membrane rafts interrupts the proximal signal cross-talk leading to subsequent NF-kappaB nuclear translocation and explains how baculovirus-expressed CD4-CDR3-like-specific rIgG(1) 13B8.2 acts to induce its biological effects.

In vitro antitumoral activity of baculovirus-expressed chimeric recombinant anti-CD4 antibody 13B8.2 on T-cell lymphomas.

A baculovirus-expressed chimeric recombinant IgG1 (rIgG1) antibody, with Cgamma1 and Ckappa human constant domains, was derived from the murine monoclonal antibody 13B8.2, which is specific for the CDR3-like loop of the CD4 molecule. The recombinant IgG1 antibody 13B8.2 was previously shown to inhibit HIV-1 replication and to abrogate the one-way mixed-lymphocyte reaction and block proliferation of CD3-stimulated peripheral blood CD4 lymphocytes from healthy donors. Before testing this recombinant anti-CD4 antibody in in vivo preclinical trials, in vitro mechanisms of action of rIgG1 13B8.2 were assessed using various CD4 T-cell lymphomas. The baculovirus-expressed rIgG1 13B8.2 antibody led to 14% to 40% proliferation inhibition of the lymphoblastic leukaemia-derived SUP-T1, the acute T lymphoma-derived CCRF-CEM and Jurkat, and the cutaneous T-Cell lymphoma-derived HUT-78 cell lines, but it did not affect the cell cycle nor induce cell apoptosis. rIgG1 antibody 13B8.2 bound the C1q fraction, leading to 9% to 17% complement-mediated lysis of the HUT-78, H9, Sup-T1, and the CCRF-CEM cell lines. No correlation was observed between cell sensitivity to rIgG1 13B8.2-triggered complement-dependent lysis and CD35-, CD46-, CD55-, and CD59-surface expression on T lymphoma cells. Using fluorescence-activated cell sorter analysis, the antibody was shown to bind to FcgammaRI/CD64-transfected IIA1.6, FcgammaRII/CD32-transfected CDw32L, and FcgammaRIII/CD16-transfected Jurkat CD16 cell lines. In correlation with these findings, rIgG1 13B8.2 induced 11% to 31% antibody-dependent cell-mediated cytotoxicity of the CCRF-CEM, SUP-T1, A2.01 CD4, and Jurkat cell lines. These convincing results on the activity of the recombinant chimeric anti-CD4 antibody 13B8.2 have led us to perform in vivo preclinical study in a murine xenograft model of CD4 lymphomas.

PIN-bodies: a new class of antibody-like proteins with CD4 specificity derived from the protein inhibitor of neuronal nitric oxide synthase.

By inserting the CB1 paratope-derived peptide (PDP) from the anti-CD4 13B8.2 antibody binding pocket into each of the three exposed loops of the protein inhibitor of neuronal nitric oxide synthase (PIN), we have combined the anti-CD4 specificity of the selected PDP with the stability, ease of expression/purification, and the known molecular architecture of the phylogenetically well-conserved PIN scaffold protein. Such "PIN-bodies" were able to bind CD4 with a better affinity and specificity than the soluble PDP; additionally, in competitive ELISA experiments, CD4-specific PIN-bodies were more potent inhibitors of the binding of the parental recombinant antibody 13B8.2 to CD4 than the soluble PDP. The efficiency of CD4-specific CB1-inserted PIN-bodies was confirmed in biological assays where these constructs showed higher potencies to block antigen presentation by inhibition of IL-2 secretion and to inhibit the one-way and two-way mixed lymphocyte reactions, compared with soluble anti-CD4 PDP CB1. Insertion of the PDP into the first exposed loop (position 33/34) of PIN appeared to be the most promising scaffold. Taken together, our findings demonstrate that the PIN molecule is a suitable scaffold to expose new peptide loops and generate small artificial ligand-binding products with defined specificities.

Biological activities on T lymphocytes of a baculovirus-expressed chimeric recombinant IgG1 antibody with specificity for the CDR3-like loop on the D1 domain of the CD4 molecule.

A baculovirus-expressed chimeric recombinant IgG1 (rIgG1) antibody, with Cgamma1 and Ckappa human constant domains, was derived from the murine monoclonal antibody (mAb) 13B8.2, which is specific for the CDR3-like loop of the CD4 molecule and which inhibits HIV-1 replication. Chimeric rIgG1 antibody 13B8.2 blocked, in a dose-dependent manner, antigen presentation through inhibition of subsequent IL-2 secretion by stimulated T cells. The one-way mixed lymphocyte reaction was abrogated by previous addition of baculovirus-produced rIgG1 13B8.2 in the T-cell culture. Anti-proliferative activity of rIgG1 was demonstrated on CD3-activated CD4+ T lymphocytes from healthy donors, such effect being associated with reduced IL-2 secretion of activated T cells. On the other hand, no proliferation inhibition was observed on CD4+ T lymphocytes activated with phorbol ester plus ionomycin, suggesting that rIgG1 13B8.2 preferentially acts on a proximal TCR-induced signaling pathway. Treatment of DBA1/J human CD4-transgenic mice with 100 microg of recombinant antibody for three consecutive days led to in vivo recovery of rIgG1 antibody 13B8.2 both coated on murine T lymphocytes and free in mouse serum, without CD4 depletion or down-modulation. These findings predict that the baculovirus-expressed chimeric rIgG1 anti-CD4 antibody 13B8.2 is a promising candidate for immunotherapy.

Mapping the paratope of anti-CD4 recombinant Fab 13B8.2 by combining parallel peptide synthesis and site-directed mutagenesis.

We analyzed antigen-binding residues from the variable domains of anti-CD4 antibody 13B8.2 using the Spot method of parallel peptide synthesis. Sixteen amino acids, defined as Spot critical residues (SCR), were identified on the basis of a 50% decrease in CD4 binding to alanine analogs of reactive peptides. Recombinant Fab 13B8.2 mutants were constructed with alanine residues in place of each of the 16 SCR, expressed in the baculovirus cell system, and purified. CD measurements indicated that the mutated proteins were conformationally intact, with a beta-sheet secondary structure similar to that of wild-type Fab. Compared with the CD4-binding capacity of wild-type Fab 13B8.2, 11 light (Y32-L, W35-L, Y36-L, H91-L, and Y92-L) and heavy chain (H35-H, R38-H, W52-H, R53-H, F100K-H, and W103-H) Fab single mutants showed a decrease in CD4 recognition as demonstrated by enzyme-linked immunosorbent assay, BIAcore, and flow cytometry analyses. The five remaining Fab mutants showed antigen-binding properties similar to those of wild-type Fab. Recombinant Fab mutants that showed decreased CD4 binding also lost their capacity to inhibit human immunodeficiency virus promoter activation and the antigen-presenting ability that wild-type Fab displays. Molecular modeling of the 13B8.2 antibody paratope indicated that most of these critical residues are appropriately positioned inside the putative CD4-binding pocket, whereas the five SCR that were not confirmed by mutagenesis show an unfavorable positioning. Taken together, these results indicate that most of the residues defined by the Spot method as critical matched with important residues defined by mutagenesis in the whole protein context. The identification of critical residues for CD4 binding in the paratope of anti-CD4 recombinant Fab 13B8.2 provides the opportunity for the generation of improved anti-CD4 molecules with more efficient pharmacological properties.