[Flow cytometry model for the detection of neutralizing antibodies against of IFN-ß]. / Modelo de detección de anticuerpos neutralizantes contra IFN-ß mediante citometría de flujo.

INTRODUCTION: Interferon beta (IFN-ß) is a treatment for relapsing remitting multiple sclerosis. However, the therapeutic use of recombinant proteins induces a humoral immunologic response resulting in the induction of binding (BAb) or neutralizing (NAb) antibodies against the biological product. The presence of neutralizing antibodies has been associated with decreased IFN-ß treatment efficacy. MATERIALS AND METHODS: Two tumor cell lines (K562 and U937) were cultivated with human recombinant IFN-ß1a at different concentrations and lengths of time in order to measure the expression of intracellular ISG15, an inducible molecule in the IFN-ß1a signaling cascade. Blood was obtained from non-immunized and IFN-ß1a immunized (100,000 IU) New Zealand rabbits. The presence of BAb was evaluated by ELISA. For NAb detection, sera 1:20 dilution were added to the IFN-ß1a-stimulated cell lines, and ISG15 expression was evaluated by flow cytometry. RESULTS: K562 cells provided the better cell line for the assay, stimulated with a dose of 1,000 IU of IFN-ß1a, and a 1:100 dilution for the primary antibody and a 1:200 dilution for the secondary antibody. ISG15 expression was compared between cells alone or cultivated with IFN-ß1a. Mean fluorescence intensity (MFI) for ISG-15 expression median was 198 arbitrary units (AU) with interquartile ranges of 173-231 AU for non-stimulated cells and 430 AU with interquartile ranges of 316-611.5 AU for IFN-ß1a stimulated cells (p<0.01). Immunized rabbit sera decreased the expression of ISG-15 in K562 cells stimulated with IFN-ß1a, whereas non-immunized rabbit sera did not. CONCLUSIONS: This rabbit model demonstrates that ISG15 expression evaluated with flow cytometry can be used as a detection assay for NAb.

Different regions of the class P-III snake venom metalloproteinase jararhagin are involved in binding to alpha2beta1 integrin and collagen.

SVMPs are multi-domain proteolytic enzymes in which disintegrin-like and cysteine-rich domains bind to cell receptors, plasma or ECM proteins. We have recently reported that jararhagin, a P-III class SVMP, binds to collagen with high affinity through an epitope located within the Da-disintegrin sub-domain. In this study, we evaluated the binding of jararhagin to alpha(2)beta(1) integrin (collagen receptor) using monoclonal antibodies and recombinant jararhagin fragments. In solid phase assays, binding of jararhagin to alpha(2)beta(1) integrin was detectable from concentrations of 20 nM. Using recombinant fragments of jararhagin, only fragment JC76 (residues 344-421), showed a significant binding to recombinant alpha(2)beta(1) integrin. The anti-jararhagin monoclonal antibody MAJar 3 efficiently neutralised binding of jararhagin to collagen, but not to recombinant alpha(2)beta(1) integrin nor to cell-surface-exposed alpha(2)beta(1) integrin (alpha(2)-K562 transfected cells and platelets). The same antibody neutralised collagen-induced platelet aggregation. Our data suggest that jararhagin binding to collagen and alpha(2)beta(1) integrin occurs by two independent motifs, which are located on disintegrin-like and cysteine-rich domains, respectively. Moreover, toxin binding to collagen appears to be sufficient to inhibit collagen-induced platelet aggregation.

Methylene blue is more toxic to erythroleukemic cells than to normal peripheral blood mononuclear cells: a possible use in chemotherapy.

Methylene blue (MB) is a phenothiazine with radio and photosensitizing properties and anti-tumoral activity. Our group has shown that MB was capable of inhibiting the in vitro growth of erythroleukemic cells with multidrug resistance (MDR). However, there are no studies comparing the cytotoxicity of this molecule for normal and tumoral cells. In this work, the cytotoxicity of MB was measured by MTT method in erythroleukemic and melanoma lineages, comparing it with that of normal cells:lymphocytes and melanocytes. MB was more cytotoxic for tumoral cells; however, there was no difference between erytroleukemic cells with or without MDR phenotype. Lymphocytes and erythroleukemic cells were much more sensitive to the effects of MB than melanoma cells and melanocytes. The proliferation of phytohemagglutinin-activated lymphocytes was inhibited when 3H-thymidine incorporation to DNA was measured. We tried to analyze whether the cells were dying, via apoptosis or necrosis, using Anexin-V and propidium iodide. Despite higher levels of Anexin-V, it was not possible to distinguish necrosis from apoptosis, as the fluorescence of MB is in the same channel as propidium iodide. The production of hydrogen peroxide was measured by cytometry using dihydrorhodamine 123 (DHR). Despite the erythroleukemic cells and lymphocytes being capable of producing free radicals, there was no relation between the production and the sensitivity of various cells to MB. Our results suggest that MB should be used as a chemotherapeutic agent, because of its preferential cytotoxic effects over tumor cells, considering the fact that MDR cells are also sensitive, and due to its radio and photosensitizing activities.

Stress-induced release of HSC70 from human tumors.

In this study, we demonstrate that the pro-inflammatory cytokine interferon-gamma (IFN-gamma) induces the active release of the constitutive form of the 70-kDa heat shock protein (HSC70) from K562 erythroleukemic cells. Treatment of K562 cells with IFN-gamma induced the upregulation of the inducible form of the 70-kDa heat shock protein (HSP70), but not the constitutive form of HSC70 within the cytosol, in a proteasome-dependent manner. In addition, IFN-gamma induced the downregulation of surface-bound HSC70, but did not significantly alter surface-bound HSP70 expression. These findings indicate that HSC70 can be actively released from tumor cells and is indicative of a previously unknown mechanism by which immune modulators stimulate the release of intracellular HSC70. This mechanism may account for the potent chaperokine activity of heat shock proteins recently observed during heat shock protein-based immunotherapy against a variety of cancers.

Bcr-Abl-mediated resistance to apoptosis is independent of constant tyrosine-kinase activity.

Bcr-Abl is one of the most potent antiapoptotic molecules and is the tyrosine-kinase implicated in Philadelphia (Ph) chromosome-positive leukemia. It is still obscure how Bcr-Abl provides the leukemic cell a strong resistance to chemotherapeutic drugs. A rational drug development produced a specific inhibitor of the catalytic activity of Bcr-Abl called STI571. This drug was shown to eliminate Bcr-Abl-positive cells both in vitro and in vivo, although resistant cells may appear in culture and relapse occurs in some patients. In the study described here, Bcr-Abl-positive cells treated with tyrosine-kinase inhibitors such as herbimycin A, genistein or STI571 lost their phosphotyrosine-containing proteins, but were still extremely resistant to apoptosis. Therefore, in the absence of tyrosine-kinase activity, Bcr-Abl-positive cells continue to signal biochemically to prevent apoptosis induced by chemotherapeutic drugs. We propose that secondary antiapoptotic signals are entirely responsible for the resistance of Bcr-Abl-positive cells. Precise determination of such signals and rational drug development against them should improve the means to combat Ph chromosome-positive leukemia.

Enhanced uptake of antisense oligonucleotides using cationic liposomes and the apoptotic effect of idarubicin in K-562 cell line.

We have evaluated the apoptotic and DNA damaging activity of Idarubicin (IDA) on K-562 cells alone and following the uptake of modified antisense-oligodeoxynucleotides (AS-ODNs) targeting b3a2 mRNA of bcr/abl hybrid gene, after treatment with AS-ODNs/DCChol-DOPE (liposomes) complexes. The uptake of FITC-labeled oligonucleotide-liposomes complexes (FITC-ODNs/DCChol-DOPE) was analyzed by flow cytometry and fluorescence microscopy. Both techniques indicated cytoplasmic accumulation of labeled liposome complexes following 24h of exposure. In absence of liposomes, AS-ODNs uptake was minimal. Pre-treatment of cells with AS-ODNs/DCChol-DOPE increased the capability of IDA to induce apoptosis as determined by morphology and the comet assay. In contrast, the use of a non-sense oligodeoxynucleotide conjugated with liposomes, in the presence of IDA, did not increase K-562 cell apoptosis. Nevertheless, DNA damage in IDA treated cells was not related to ODNs/liposomes pre-treatment, as determined by the comet assay. Our data suggests that DCChol-DOPE increases the uptake of ODNs in K-562 cells, and these modified AS-ODNs increase IDA induced apoptosis by decreasing p210(bcr/abl) levels in K-562 cells.

Direct current decreases cell viability but not P-glycoprotein expression and function in human multidrug resistant leukemic cells.

Inhibition of tumor growth induced by treatment with direct current (DC) has been reported in several systems. In the current work, the cellular effects generated by the DC treatment of the human leukemic K562 cell line and its vincristine-resistant derivative K562-Lucena 1 were analyzed by trypan blue staining and transmission electron microscopy. DC stimulation induced cell lysis, alterations in shape, membrane extraction or discontinuity, and intense vacuolization of some cells. In addition, treatment of K562 and K562-Lucena 1 cells caused a marked decrease in viability. Since multidrug resistance is a major factor contributing with failure of chemotherapy in many tumors, the expression and function of P-glycoprotein (P-gp) in K562-Lucena 1 cells were also studied. The expression of mdr1, the gene encoding P-gp, was analyzed by reverse transcription polymerase chain reaction, which showed that this gene was equally expressed in either treated or untreated cells. These results were confirmed by flow cytometry with a monoclonal anti P-gp antibody and the Rhodamine 123 extrusion method, which revealed that P-gp surface expression and function were unaltered after DC treatment. Our results suggest that DC treatment does not affect P-gp in human leukemic cells, but affects their viability by mechanisms that would involve clear cellular effects, but also additional targets, whose relevance in dc treated tumoral cells is currently discussed.