Evaluation of Elecsys Syphilis Assay for Routine and Blood Screening and Detection of Early Infection.

Treponema pallidum infections can have severe complications if not diagnosed and treated at an early stage. Screening and diagnosis of syphilis require assays with high specificity and sensitivity. The Elecsys Syphilis assay is an automated treponemal immunoassay for the detection of antibodies against T. pallidum The performance of this assay was investigated previously in a multicenter study. The current study expands on that evaluation in a variety of diagnostic settings and patient populations, at seven independent laboratories. The samples included routine diagnostic samples, blood donation samples, samples from patients with confirmed HIV infections, samples from living organ or bone marrow donors, and banked samples, including samples previously confirmed as syphilis positive. This study also investigated the seroconversion sensitivity of the assay. With a total of 1,965 syphilis-negative routine diagnostic samples and 5,792 syphilis-negative samples collected from blood donations, the Elecsys Syphilis assay had specificity values of 99.85% and 99.86%, respectively. With 333 samples previously identified as syphilis positive, the sensitivity was 100% regardless of disease stage. The assay also showed 100% sensitivity and specificity with samples from 69 patients coinfected with HIV. The Elecsys Syphilis assay detected infection in the same bleed or earlier, compared with comparator assays, in a set of sequential samples from a patient with primary syphilis. In archived serial blood samples collected from 14 patients with direct diagnoses of primary syphilis, the Elecsys Syphilis assay detected T. pallidum antibodies for 3 patients for whom antibodies were not detected with the Architect Syphilis TP assay, indicating a trend for earlier detection of infection, which may have the potential to shorten the time between infection and reactive screening test results.

Generation of rabbit antibodies against death ligands by cDNA immunization.

Specificity problems, especially in immunoblot analysis, have been shown for several commercial antibodies raised against the death ligand Fas ligand (FasL) using conventional protein and/or peptide immunizations. In this work, we have optimized the development of rabbit antisera and isolated pAb against the death ligands FasL, Apo2 ligand/TRAIL and Apo3 ligand/TWEAK by cDNA intramuscular immunization. This alternative approach has generated specific pAb in all three cases, which are useful for immunoblot purposes. The present data suggest that for the production of antibodies against certain glycosylated membrane proteins, cDNA immunization could be the method of choice.

Differential secretion of Fas ligand- or APO2 ligand/TNF-related apoptosis-inducing ligand-carrying microvesicles during activation-induced death of human T cells.

Preformed Fas ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL) are stored in the cytoplasm of the human Jurkat T cell line and of normal human T cell blasts. The rapid release of these molecules in their bioactive form is involved in activation-induced cell death. In this study, we show by confocal microscopy that FasL and APO2L/TRAIL are mainly localized in lysosomal-like compartments in these cells. We show also by immunoelectron microscopy that FasL and APO2L/TRAIL are stored inside cytoplasmic compartments approximately 500 nm in diameter, with characteristics of multivesicular bodies. Most of these compartments share FasL and APO2L/TRAIL, although exclusive APO2L/TRAIL labeling can be also observed in separate compartments. Upon PHA activation, the mobilization of these compartments toward the plasma membrane is evident, resulting in the secretion of the internal microvesicles loaded with FasL and APO2L/TRAIL. In the case of activation with anti-CD59 mAb, the secretion of microvesicles labeled preferentially with APO2L/TRAIL predominates. These data provide the basis of a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation and could modify the interpretation of the role of FasL and APO2L/TRAIL as effector mechanisms in physiological and pathological situations.

Remodelling of the actin cytoskeleton is essential for replication of intravacuolar Salmonella.

Maturation and maintenance of the intracellular vacuole in which Salmonella replicates is controlled by virulence proteins including the type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2). Here, we show that, several hours after bacterial uptake into different host cell types, Salmonella induces the formation of an F-actin meshwork around bacterial vacuoles. This structure is assembled de novo from the cellular G-actin pool in close proximity to the Salmonella vacuolar membrane. We demonstrate that the phenomenon does not require the Inv/Spa type III secretion system or cognate effector proteins, which induce actin polymerization during bacterial invasion, but does require a functional SPI-2 type III secretion system, which plays an important role in intracellular replication and systemic infection in mice. Treatment with actin-depolymerizing agents significantly inhibited intramacrophage replication of wild-type Salmonella typhimurium. Furthermore, after this treatment, wild-type bacteria were released into the host cell cytoplasm, whereas SPI-2 mutant bacteria remained within vacuoles. We conclude that actin assembly plays an important role in the establishment of an intracellular niche that sustains bacterial growth.

Unusual intracellular trafficking of Salmonella typhimurium in human melanoma cells.

Salmonella spp. are enterobacteria capable of invading and replicating in both professional and non-professional phagocytes. Here, we investigate the fate of S. typhimurium in human melanoma MelJuSo cells. The bacterium entered MelJuSo cells by a trigger mechanism and resided within a unique organelle, the Salmonella-containing vacuole (SCV). The SCV acquired early endosomal markers transiently and then underwent a series of membrane modifications. In HeLa cells, vacuole maturation is characterized by the simultaneous acquisition of the lysosomal membrane glycoproteins (Lgps) Lamp1, CD63 and vacuolar (v)-ATPase; in MelJuSo cells, however, acquisition of CD63 and v-ATPase preceded that of Lamp1. A very striking event in MelJuSo cells was the arrest of bacterial septation starting from 8 h after infection. Bacteria nevertheless continued to elongate, remained morphologically intact and viable and were eventually exocytosed. This original feature was observed in several skin-related cells including melanocytes, suggesting that it may provide the basis for an efficient host defence mechanism against Salmonella infection.

Essential role of the VirB machinery in the maturation of the Brucella abortus-containing vacuole.

In epithelial cells, the intracellular pathogen Brucella abortus escapes from the endocytic pathway, exploits the autophagic machinery of the host cell and establishes a unique replication niche in the endoplasmic reticulum. The molecular mechanisms underlying these processes are still poorly understood. Recently, a B. abortus type IV-related secretion system encoded by the virB operon has been described as being involved in the intracellular trafficking of the bacteria. In this study, we have analysed the intracellular pathway of B. abortus virB10 mutant strains by confocal microscopy. We demonstrate that a functional virB operon is essential for the biogenesis of the Brucella-containing vacuole. Polar mutation preventing the transcription of virB10 and downstream sequences did not allow Brucella to bypass the endocytic pathway. Consequently, polar mutant-containing vacuoles fused with lysosomes in which bacteria underwent a degradation process. In contrast, virB10 non-polar mutants were capable of avoiding interactions with the endocytic pathway but, diverging to wild-type Brucella, were unable to reach the endoplasmic reticulum to establish their intracellular replication niche and seemed to be recycled to the cell surface. Based on the two particular phenotypes described in this work, a model of maturation of the Brucella-containing vacuole is proposed.

Tyrosine phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase correlates with high proliferation rates in sublines derived from the Jurkat leukemia.

A prominent tyrosine phosphorylated protein of 85 kDa (p85) was detected in highly proliferative sublines derived from the Jurkat T cell leukemia. We undertook a study to characterize the identity of this protein and its possible role in the hyperproliferative phenotypes observed. Using immunoblot and immunoprecipitation techniques, this protein was characterized as the p85 regulatory subunit of phosphatidylinositol 3-kinase. Cell proliferation and p85 tyrosine phosphorylation was not affected by tyrphostin AG-490, an inhibitor of Jak kinases, wortmannin or LY294002, inhibitors of the activity of the catalytic phosphatidylinositol 3-kinase subunit. Herbimycin-A and PPI, inhibitors of src-like protein tyrosine kinases, and genistein, a general tyrosine kinase inhibitor, inhibited p85 tyrosine phosphorylation and induced cell death in the sublines. PD98059, an inhibitor of Mek, inhibited cell growth of the sublines, but not that of the parental cells. It was concluded that tyrosine phosphorylation of p85 is associated with highly proliferative tumoral phenotypes, at least in T cell leukemias, independent of the phosphatidylinositol 3-kinase activity of the catalytic subunit.

CD59 cross-linking induces secretion of APO2 ligand in overactivated human T cells.

Jurkat cells and the derived TCR / CD3-defective subline, J.RT3.T3.5 undergo activation induced cell death (AICD) when stimulated with phytohemagglutinin (PHA). Since J.RT3.T3.5 cells do not express antigen receptor, we searched for the molecules that could be ligated by PHA and induce AICD in this cell line. We show here that the glycosylphosphatidylinositol linked CD59 molecule is expressed at the surface of Jurkat and J.RT3.T3.5 cells, and when cross-linked by specific antibodies can induce cell death. The toxicity of supernatants from PHA-stimulated Jurkat or J.RT3.T3.5 cells was prevented by a combination of the blocking anti-Fas mAb SM1 / 23 and anti-APO2L / TRAIL mAb 5C2. However, toxicity of supernatants from anti-CD59 stimulated cells was specifically prevented by the anti-APO2L blocking antibody. Anti-CD59 cross-linking induced AICD also in normal human T cell blasts, which secreted toxic molecules into the supernatant. The toxicity of these supernatants on Jurkat cells was fully prevented by the anti-APO2L blocking antibody, showing that CD59 crosslinking induces the preferential release of APO2L also in normal T cells. The possible physiological and / or pathological consequences of this observation are discussed.

Activated human T cells release bioactive Fas ligand and APO2 ligand in microvesicles.

Activation-induced cell death is a process by which overactivated T cells are eliminated, thus preventing potential autoimmune attacks. Two known mediators of activation-induced cell death are Fas(CD95) ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL). We show here that upon mitogenic stimulation, bioactive FasL and APO2L are released from the T cell leukemia Jurkat and from normal human T cell blasts as intact, nonproteolyzed proteins associated with a particulate, ultracentrifugable fraction. We have characterized this fraction as microvesicles of 100-200 nm in diameter. These microvesicles are released from Jurkat and T cell blasts shortly (

Involvement of APO2 ligand/TRAIL in activation-induced death of Jurkat and human peripheral blood T cells.

The interaction of Fas with Fas ligand (FasL) mediates activation-induced cell death (AICD) of T hybridomas and of mature T lymphocytes. The TNF/TNF receptor system also plays a significant role in AICD of mature T cells and in the maintenance of peripheral tolerance. We previously demonstrated that in human Jurkat leukemia cells, AICD is triggered mainly by the rapid release of preformed FasL upon TCR stimulation. In the present work, we show that the cytotoxic cytokine APO2 ligand (APO2L; also known as TRAIL) is constitutively expressed as an intracytoplasmic protein in Jurkat T cells and derived sublines. APO2L is also detected in fresh human peripheral blood mononuclear cells (PBMC) from a significant number of donors, and the amount of both FasL and APO2L substantially increases upon blast generation. A neutralizing anti-APO2L monoclonal antibody (mAb) partially suppresses the cytotoxicity induced by supernatants of phytohemagglutinin (PHA)-prestimulated Jurkat or human PBMC on non-activated Jurkat cells, indicating that APO2L is released by these cells and contributes to AICD. A combination of neutralizing anti-APO2L and anti-Fas mAb blocks around 60 % of the toxicity associated with supernatants from PHA-activated human PBMC. These results show that FasL and APO2L account for the majority of cytotoxic activity released during AICD, and suggest that additional uncharacterized factors may also contribute to this process.

Resistance to apoptosis correlates with a highly proliferative phenotype and loss of Fas and CPP32 (caspase-3) expression in human leukemia cells.

Apoptosis induced by effector cells of the immune system or by cytotoxic drugs is a main mechanism mediating the prevention or elimination of tumoral cells. For instance, the human T-cell leukemia Jurkat is sensitive to Fas-induced apoptosis and to activation-induced cell death (AICD), and the promonocytic leukemia U937 is sensitive to Fas- and TNF-induced apoptosis. In this work, we have analyzed the mechanisms of resistance to physiological or pharmacological apoptosis in human leukemia by generating highly proliferative (hp) sub-lines derived from Jurkat and U937 cells. These hp sub-lines were resistant to Fas- and TNF-induced apoptosis, as well as to AICD. This was due to the complete loss of Fas and TNFR surface expression and, in the case of Jurkat-derived sub-lines, also of CD3, CD2 and CD59 molecules. The sub-lines also completely lacked the expression of the apoptotic protease CPP32, present in parental cells. Moreover, these sub-lines were no longer sensitive to doxorubicin-induced apoptosis, which was efficiently blocked by the general caspase inhibitor Z-VAD-fmk in the parental cell lines. These data suggest a molecular mechanism for the development of resistance of leukemic cells to physiological and pharmacological apoptosis inducers, giving rise to highly proliferative tumoral phenotypes. These results also indicate that Fas and CPP32 could be useful prognostic markers for the progression and/or therapy outcome of human leukemias.

Doxorubicin-induced apoptosis in human T-cell leukemia is mediated by caspase-3 activation in a Fas-independent way.

It has recently been proposed that doxorubicin (DOX) can induce apoptosis in human T-leukemia cells via the Fas/FasL system in an autocrine/paracrine way. We show here that treatment of Jurkat cells with either anti-Fas antibodies, anthracyclin drugs or actinomycin D induces the activation of CPP32 (caspase-3) and apoptosis. However, DOX treatment did not induce the expression of membrane FasL or the release of soluble FasL and co-incubation with blocking anti-Fas antibodies prevented Fas-induced but not DOX-induced apoptosis. All the morphological and biochemical signs of apoptosis induced by anti-Fas or DOX can be prevented by Z-VAD-fmk, a general caspase inhibitor. DEVD-cho, a specific inhibitor of CPP32-like caspases which completely blocks Fas-mediated apoptosis, prevented drug-induced nuclear apoptosis but not cell death. We conclude that: (i) DOX-induced apoptosis in human T-leukemia/lymphoma is Fas-independent and (ii) caspase-3 is responsible of DOX-induced nuclear apoptosis but other Z-VAD-sensitive caspases are implicated in cell death.

Influence on CD8 of TCR/CD3-generated signals in CTL clones and CTL precursor cells.

Alloreactive CTL clones and naive CTL precursor cells (CTLp) from TCR-transgenic mice were analyzed for their response in total and in TCR-associated kinase activation upon stimulation with the relevant class I allo-APCs. The responses were found to be stronger and more sustained for the CTL clone and CTLp expressing a TCR previously characterized as CD8 coreceptor independent than for the CTL clone and CTLp expressing a TCR characterized as CD8 dependent. Unexpectedly, it was found that also in response to CD3 engagement, total and TCR-associated kinase activation were stronger and more sustained in the CTL clone and CTLp expressing the CD8-independent TCR. In both types of CTL clones, p56(lck) was found associated with the TCR complex, and CD3 components were found associated with CD8 before CD3 engagement. Upon CD3 engagement, ZAP-70 was also found associated with the TCR complex and the kinase activity (p56(lck)) associated with CD8 increased. This increase was more pronounced for the CD8-independent than for the CD8-dependent clone. An increased association of CD3zeta with CD8 was also detected after CD3 engagement for each clone. These data indicate that signals resulting from exclusive CD3 engagement can influence CD8 molecular associations and activate CD8-bound p56(lck). They further suggest that clonal differences exist that influence the efficiency of signaling upon binding of the same CD3 ligand. The observation that this property was shared between independently derived CTL clone and CTLp expressing the same TCR suggests that it may be acquired during repertoire selection.

Release of preformed Fas ligand in soluble form is the major factor for activation-induced death of Jurkat T cells.

Interaction of Fas/APO-1 (CD95) and its ligand (FasL) plays an important role in the activation-induced cell death (AICD) of T lymphocytes. In the present work, the contribution of soluble FasL to AICD of the human T-cell line Jurkat has been studied. Jurkat cells prestimulated with phytohaemagglutinin (PHA) induced the death of non-activated Jurkat cells, and also of L1210Fas, but not that of Fas-negative L1210 cells. Culture supernatants from prestimulated Jurkat cells were highly toxic to their non-activated counterparts. Time-course analysis revealed that PHA-stimulated Jurkat cells quickly release (less than 15 min) to the medium a toxic molecule following a biphasic pattern, with maximal cytotoxic activities at 1 hr and 7 hr after stimulation. The cytotoxic effect of those supernatants was prevented by the addition of a blocking anti-Fas monoclonal antibody, suggesting that PHA-stimulated Jurkat cells exert Fas-based cytotoxicity mainly through the release of soluble FasL. The constitutive intracellular expression of FasL in non-activated Jurkat cells and its release as a consequence of PHA activation were detected by immunostaining and immunoblotting using an anti-FasL antibody. These data indicate that, at least in Jurkat cells, AICD is mainly mediated by the rapid release of performed FasL in soluble form upon stimulation.

Biosynthesis of unsaturated fatty acids in the main cell lineages of human leukemia and lymphoma.

Unsaturated fatty acids are essential for the proliferation of many haematopoietic cells, but little is known about their biosynthetic pathways in these cells. We have studied the activity of the main desaturation-elongation enzymes in human B-(Reh-6, Raji, Ramos) and T-(CEM, Jurkat) lymphocytic, promonocytic (U937), promyelocytic (HL-60) and pluripotent myeloid (K562) cell lineages, as well as the changes induced by cell differentiation. Cells were incubated with 14C-labelled 18:0, 18:2(n - 6) and 18:3(n - 3) or supplemented with the corresponding unlabelled fatty acid and synthesis of polyunsaturated fatty acids (PUFA) was evaluated by argentation-TLC and GLC. The main activity present in most cells was delta 9-desaturase (range between 200-1000 pmol/24 h per 10(6) cells) that was regulated by the type of free fatty acids in culture media. A great variability in the activities of delta 6- and delta 5-desaturase was observed. They were virtually absent in B-cells and only one (Jurkat) T-cell line synthesized significant amounts of (n - 6) and (n - 3) PUFA. The main PUFA formed by Jurkat cells were 20:3 and 20:4(n - 6) (30 and 40%, respectively, of cell lipid radioactivity) and 20:5, 22:5 and 22:6(n - 3) (60, 20 and 10%, respectively, of cell radioactivity). Cell differentiation caused complex changes in desaturase activities. The activity of delta 9-desaturase increased with the degree of differentiation of B-cells. Differentiation of U937 cells to macrophages with PMA caused a 2-3-fold increase in the activity of (delta 6 + delta 5)- and delta 9-desaturases and no changes and a 2-fold decrease, respectively, if the inducer was DMSO. Differentiation of HL-60 cells to granulocytes with DMSO virtually abolished delta 9-desaturase activity and greatly reduced that of delta 6- and delta 5-desaturases. delta 9-Desaturase activity increased (2.5-fold) in myeloid K562 cells differentiated to erythroblasts with hemin. No induction of delta 6-desaturase, absent in K562 cells, occurred after differentiation to erythroblasts or megakaryoblasts and they synthesized alternative PUFA through sequential elongation and delta 5-desaturation of 18:2(n - 6) and 18:3(n - 3). The activities of delta 6- and delta 5-desaturase in HL-60 and U937 cells increased when differentiation also stimulated the synthesis of eicosanoids and extracellular release of PUFA.

Alternative route for the biosynthesis of polyunsaturated fatty acids in K562 cells.

K562 human leukaemia cells lack a significant delta 6-desaturase activity. However, they synthesize long-chain polyunsaturated fatty acids (PUFA) from linoleic (C18:2(9,12)) and linolenic (C18:3(9,12,15)) acids, by reactions involving a C2 chain elongation followed by a delta 5-desaturation step and, to some extent, a further elongation. The main products formed were separated by argentation t.l.c. and identified by g.l.c. as the uncommon fatty acids C20:3(5,11,14) and C20:4(5,11,14,17) respectively. These acids were also produced when cells were supplemented with C20:2(11,14) or C20:3(11,14,17) respectively. The presence of a delta 5-desaturase was further confirmed by using its corresponding normal substrates, C20:3(8,11,14) and C20:4(8,11,14,17), which led to C20:4(5,8,11,14) and C20:5(5,8,11,14,17) respectively. On the other hand, a high delta 9-desaturase activity, but no significant delta 4-desaturase activity, were detected in K562 cells. These results indicate the existence of an alternative pathway, involving delta 5-desaturase, which is the only route for PUFA biosynthesis in K562 cells. This pathway may be relevant for the biosynthesis of PUFA in cells lacking delta 6-desaturase activity.