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The article focuses on a study which aims to investigate the profile of microvesicles (MV) in myelofibrosis (MF) and essential thrombocythaemia (ET) whether MV proportions could be related to severity of MF as well as the role of inflammation on MV frequency in MF. Information about the effects of ruxolitinib on MVs in MF and the frequency of circulating megakaryocyte- and platelet-MVs is presented.

B Introduction: b Immune effector cell-associated neurotoxicity syndrome (ICANS) is a life-threatening adverse effect of anti-CD19 chimeric antigen receptor (CAR) T-cell therapy that usually occurs within 5-7 days after cell infusion. Predicting ICANS by means of plasma CAR-T cell derived extracellular vesicles in patients undergoing infusion of anti-CD19 CAR-T cells Twenty out of 71 patients (28%) had ICANS of any grade: 5 patients (7%) ICANS grade 1, 7 patients (10%) ICANS grade 2 and 8 patients (11%) ICANS grade >=3 (3 patients ICANS grade 3, 3 patients ICANS grade 4 and 2 patients ICANS grade 5 with diffuse cerebral edema). [Extracted from the article]

Electrospray Ionization and collision induced dissociation tandem mass spectrometry are usually employed to obtain compound identification through a mass spectra match. Different algorithms have been developed for this purpose (for example the NIST match algorithm). These approaches compare the tandem mass spectra of the unknown analyte with the tandem mass spectra spectra of known compounds inserted in a database. The compounds are usually identified on the basis of spectral match value associated with a probability of recognition. However, this approach is not usually applied to multiple reaction monitoring transition spectra achieved bymeans of triple quadrupole apparatus, mainly due to the lack of a transition spectra database. The Surface Activated Chemical Ionization-Electrospray-NIST Bayesian model database search (SANIST) platform has been recently developed for new potential metabolite biomarker discovery, to confirm their identity and to use themfor clinical and diagnostic applications. Here, we present an improved version of the SANIST platform that extends its application to forensic, pharmaceutical, and food analysis studies, where the compound identification rules are strict. The European Union (EU) has set directives for compound identification (EU directive 2002/657/EC). We have applied the SANIST method to identification of 11-nor-9-carboxytetrahydro-cannabinol in urine samples (an example of a forensic application), circulating levels of the immunosuppressive drug tacrolimus in blood (an example of a pharmaceutical application) and glyphosate in fruit juice (an example of a food analysis application) that meet the EU directive requirements. [ABSTRACT FROM AUTHOR]

Background: The presence of antibodies to CTLA-4, a negative regulator of T-cell activation, was investigated in multiply transfused patients with malignant and non- malignant hematologic diseases. A previous study showed that, in multiply transfused patients, an immune response against nuclear matrix proteins can be induced by WBCs undergoing apoptosis during RBC unit storage. This study evaluated whether the same phenomenon could be involved in the induction of CTLA-4 antibodies in the patients analyzed.Study Design and Methods: Patient sera were tested for binding to the recombinant full-length CTLA-4 beta-galactosidase fusion protein by an ELISA. Immuno-fluorescence stainings were performed to analyze the CTLA-4 epitopes recognized by the antibodies and to detect such epitopes in the apoptotic cells present in the RBC units.Results: CTLA-4 antibodies were found in multiply transfused patients with beta-thalassemia (40%) and with other hemolytic diseases (33%) including leukemias (42%). A higher incidence of CTLA-4 antibodies was found in patients receiving non-WBC-reduced blood (88%) than in those receiving WBC-reduced blood (26%). Immunofluorescence staining showed that WBCs undergoing apoptosis in the RBC unit expressed CTLA-4 epitopes.Conclusions: The apoptotic WBCs present in the RBC units, after cold storage, express CTLA-4 epitopes. These epitopes can be released and induce formation of CTLA-4 antibodies with profound implications in the development of autoimmune disorders and in facilitating tumor dissemination and metastasis. [ABSTRACT FROM AUTHOR]

Background and Objectives The haemoglobin level of prospective blood donors is usually performed on blood obtained by from the finger pulp by fingerstick with a lancet and filling a capillary tube with a sample. New noninvasive methods are now available for rapid, noninvasive predonation haemoglobin screening. Materials and Methods Prospective blood donors at our blood centre were tested, in two different trials, as follows: by the NBM 200 ( Or Sense) test ( n = 445 donors) and by the Pronto-7 ( Masimo) test ( n = 463 donors). The haemoglobin values of each trial and the haemoglobin of finger pulp blood obtained by fingerstick with a lancet ( Hemo Cue) were compared with the haemoglobin values obtained from a venous sample on a Cell Counter ( Beckman Coulter). Results Comparison of Beckman Coulter Cell Counter and Or Sense and results showed a bias of 0·29 g/dl, the standard deviation of the differences ( SDD) of 0·98 and 95% limits of agreement from −1·64 to 2·21, using Bland and Altman statistical methodology. Comparison of Masimo and Beckman Coulter Cell Counter results showed a bias of −0·53 g/dl, SDD of 1·04 and 95% limits of agreement from −2·57 to 1·51. Cumulative analysis of all 908 donors, as tested by the usual fingerstick test showed a bias of 0·83 g/dl, SDD of 0·70 and 95% limits of agreement from −0·54 to 2·20 compared with the Coulter Cell Counter. Compared with the Coulter Counter, the specificity of the methods was 99·5% for fingerstick, 97% for Or Sense and 83% for Massimo, and the sensitivity was 99, 98 and 93%, respectively. Conclusions Analysis of finger pulp blood by either direct sampling by fingerstick and Hemocue, or by noninvasive haemoglobin tests does not replicate the results of cell counter analysis of venous samples. Compared with fingerstick, noninvasive haemoglobin tests eliminate pain and reduce stress, but have a lower level of specificity and sensitivity. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate whether mesenchymal stem cells (MSC) and platelet lysate (PL) seeded in a fibrin or collagen scaffold could improve the new bone (NB) formation around an uncemented hip prosthesis stem in a sheep model. In vitro expanded MSC were suspended in PL and either mixed with collagen or fibrin gel as delivery vehicle. The cell-gel composites were inserted inside the femoral canal, then the prosthesis was press-fit inserted inside the femur. Identical procedures were performed in a control group, but only the prosthesis was implanted. Histomorphometrical analysis performed 4 months after surgery indicated that the newly formed bone inside the medullary canal, between the inner cortex and the prosthetic stem, was significantly higher in the MSC-PL-collagen group (mean 18.7 ± 4.5%) and in the MSC-PL-fibrin group (mean 18.8 ± 15.2%) when compared to the control group (mean 4.6 ± 2.0%). There was a significantly higher bone-prosthesis contact in the MSC-PL-collagen group (mean 2.7 ± 2.6%) and in the MSC-PL-fibrin group (mean 2.3 ± 3.1%) compared to the control group (mean 0.2 ± 0.1%). The results indicate that MSC and PL in a fibrin or collagen scaffold can promote NB formation around an uncemented hip prosthesis stem. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:961-968 [ABSTRACT FROM AUTHOR]

Over the past 20 years, survival rates of T-cell acute lymphoblastic leukemia (T-ALL) patients have improved, mainly because of advances in polychemotherapy protocols. Despite these improvements, we still need novel and less toxic treatment strategies targeting aberrantly activated signaling networks which increase proliferation, survival, and drug resistance of T-ALL cells. One such network is represented by the phosphatidylinositol 3-kinase (PI3K)/Akt axis. PI3K inhibitors have displayed some promising effects in preclinical models of T-ALL. Here, we have analyzed the therapeutic potential of the Akt inhibitor, triciribine, in T-ALL cell lines. Triciribine caused cell cycle arrest and caspase-dependent apoptosis. Western blots demonstrated a dose-dependent dephosphorylation of Akt1/Akt2, and of mammalian target of rapamycin complex 1 downstream targets in response to triciribine. Triciribine induced autophagy, which could be interpreted as a defensive mechanism, because an autophagy inhibitor (chloroquine) increased triciribine-induced apoptosis. Triciribine synergized with vincristine, a chemotherapeutic drug employed for treating T-ALL patients, and targeted the side population of T-ALL cell lines, which might correspond to leukemia initiating cells. Our findings indicate that Akt inhibition, either alone or in combination with chemotherapeutic drugs, may serve as an efficient treatment towards T-ALL cells requiring upregulation of this signaling pathway for their proliferation and survival. J. Cell. Physiol. 226: 822-831, 2011. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Anti-thymocyte globulins (ATG) are currently used to prevent graft- versus-host disease in haematopoietic stem cell transplants from alternative donors and to treat and prevent acute organ rejection after transplantation. Many recent studies have demonstrated that ATG can also be beneficial in patients with myeloma, lymphoma, leukaemia and myelodysplastic syndrome. This study showed, for the first time, that the cytotoxic effect of ATG can been enhanced by conjugation with saporin-S6, which is one of the most stable and active type-1 ribosome-inactivating proteins. The ATG-saporin-S6 immunotoxin showed a strong cytotoxic effect on five lymphoma- and leukaemia-derived cell lines as well as on activated lymphocytes while sparing non-haematological cell lines. ATG-saporin-S6 induced a time-dependent activation of caspase-3/7 in RAJI cells. The caspase inhibitor Z-VAD-fmk partially rescued the cells that were treated with ATG-saporin-S6, suggesting that multiple cell death pathways, some of which are caspase independent, play a role in ATG-saporin-S6 toxicity. In our experiments ATG increased the complement-independent cytotoxicity of activated lymphocytes by a magnitude of 3–5 logs after conjugation. These findings suggest that the ATG-saporin-S6 immunotoxin is a promising therapeutic tool for many pathological conditions involving T lymphocytes and T and B neoplastic cells. [ABSTRACT FROM AUTHOR]

OBJECTIVE To study the effect of the H2S-donating derivative of sildenafil (ACS6) compared to sildenafil citrate and sodium hydrosulphide (NaHS) on relaxation, superoxide formation and NADPH oxidase and type 5 phosphodiesterase (PDE5) expression in isolated rabbit cavernosal tissue and smooth muscle cells (CSMCs), and in vivo on indices of oxidative stress induced with buthionine sulphoximine (BSO). MATERIALS AND METHODS Relaxation was studied in an organ bath in response to carbachol and after incubation with interleukin-1β for 12 h. CSMCs were incubated with tumour-necrosis factor-α or the thromboxane A2 (TXA2) analogue, U46619, with or with no sildenafil citrate, ACS6 or NaHS for 16 h. Superoxide formation and the expression of p47phox (an active subunit of the NADPH oxidase complex) and PDE5 protein was then assessed using Western blotting. Rats were also treated with BSO (with or with no sildenafil citrate or ACS6) for 7 days; cavernosal cGMP, cAMP, glutathionine and plasma TXA2 and 8-isoprostane F2α was measured by enzyme-linked immunosorbent assay. RESULTS ACS6 and sildenafil citrate relaxed cavernosal smooth muscle equipotently; NaHS alone had little effect at up to 100 µm. The formation of superoxide and expression of p47phox and PDE5 was reduced by ACS6, sildenafil citrate and NaHS (order of potency: ACS6 > sildenafil citrate > NaHS). The effects of ACS6 were blocked by inhibitors of protein kinase A (PKA) and PKG. In rats treated with BSO, both ASC6 and sildenafil citrate reduced the increased plasma levels of TXA2 and 8-isoprostane F2α but increased cGMP, cAMP and glutathionine levels in corpus cavernosum. CONCLUSIONS By virtue of a dual action on PKA and PKG activation, ACS6 not only promotes erection, acutely, but might also have a long-term beneficial effect through inhibition of oxidative stress and downregulation of PDE5. [ABSTRACT FROM AUTHOR]

Background and purpose:Hydrogen sulphide (H2S) is an endogenous gaseous mediator active in the multilevel regulation of pathophysiological functions in mammalian cardiovascular tissues.Experimental approach:This study investigated the pharmacological activity of a new H2S-releasing derivative of diclofenac, S-diclofenac (2-[(2,6-dichlorophenyl)amino]benzeneacetic acid 4-(3H-1,2-dithiole-3-thione-5-yl)-phenyl ester) in the isolated rabbit heart submitted to low-flow ischaemia-reperfusion damage.Key results:S-diclofenac (3, 10 and 30 μM), despite inhibiting prostacyclin generation by cardiac tissues, achieved dose-dependent normalization of coronary perfusion pressure, reducing left ventricular contracture during ischaemia and improving left ventricular developed pressure and ±dP/dtmax at reperfusion. Creatine kinase and lactate dehydrogenase activities in heart perfusates were significantly reduced during reperfusion. These effects were accompanied by substantial release of reduced glutathione (GSH), indicating that the H2S moiety may have up-regulated cysteine transport. The anti-ischaemic activities of S-diclofenac and the H2S-donor sodium hydro sulphide (NaHS) were partially prevented by the KATP channel antagonist glibenclamide, suggesting a mechanism similar to H2S-induced cardioprotection in metabolic ischaemic preconditioning. Perfusion with the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine worsened the myocardial ischaemia-reperfusion damage, but this was dose-dependently prevented by S-diclofenac and NaHS, suggesting that the released H2S may have overcome NO deficiency.Conclusion and implications:These data show that S-diclofenac had marked anti-ischaemic activity in ischaemic-reperfused rabbit hearts despite inhibition of prostaglandin generation. Increased GSH formation leading to activation of KATP channels may have contributed to this beneficial effect. The pharmacological profile of S-diclofenac and its anti-inflammatory activity, with diminished gastrointestinal side effects, offer therapeutic applications in cardiovascular disease.British Journal of Pharmacology (2008) 153, 100–109; doi:10.1038/sj.bjp.0707540; published online 29 October 2007 [ABSTRACT FROM AUTHOR]

The nucleus contains diacylglycerol kinases (DGKs), i.e., the enzymes that, by converting diacylglycerol (DG) into phosphatidic acid, terminate DG-dependent events. It has been demonstrated that nuclear DGK-ζ interferes with cell cycle progression. We previously reported that nuclear DGK-ζ expression increased during myogenic differentiation, whereas its down-regulation impaired differentiation. Here, we evaluated the possible involvement of nuclear DGK-ζ in cell cycle progression of C2C12 myoblasts. Overexpression of a wild-type DGK-ζ, which mainly localized to the nucleus (but not of a kinase dead mutant or of a mutant that did not enter the nucleus), blocked the cells in the G1 phase of the cell cycle, as demonstrated by in situ analysis of biotinylated-l6-dUTP incorporated into newly synthesized DNA and by flow cytometry. In contrast, down-regulation of endogenous DGK-ζ by short interfering RNA (siRNA) increased the number of cells in both the S and G2/M phases of the cell cycle. Cell cycle arrest of cells overexpressing wild-type DGK-ζ was accompanied by decreased levels of retinoblastoma protein phosphorylated on Ser-807/811. Down-regulation of endogenous DGK-ζ, using siRNA, prevented the cell cycle block characterizing C2C12 cell myogenic differentiation. Overall, our results identify nuclear DGK-ζ as a key determinant of cell cycle progression and differentiation of C2C12 cells. [ABSTRACT FROM AUTHOR]

We have previously reported that about 80% of acute myeloid leukaemia (AML) samples tested at diagnosis constitutively expressed cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). The present study compared CTLA-4 expression and function of leukaemic cells from AML patients at diagnosis with those from AML patients resistant to conventional chemotherapy. We also explored the possibility of targeting CTLA-4 for apoptosis induction in chemoresistant AML cells. AML cells either from untreated patients ( n = 15) or in chemoresistant phase ( n = 10) were analysed for CTLA-4 protein and transcript expression by flow cytometry and reverse transcription-polymerase chain reaction respectively. CTLA-4 expression was similar in untreated and in chemoresistant samples and was not associated with patients’ clinical features. In chemoresistant AML cells, CTLA-4 transduced an apoptotic signal on engagement with its recombinant ligands r-CD80 and r-CD86, which induced an average of 71% and 62% apoptotic cells, respectively, at highest concentration. Apoptosis was equally induced in untreated leukaemic cells accompanied by cleavage of procaspase-8 and -3. Thus, this study provides the first evidence that killing of leukaemic cells from AML patients may be obtained by the engagement of CTLA-4 with its ligands, opening the way to a novel potential therapeutic approach based on triggering the CTLA-4 molecule to circumvent chemoresistance in AML. [ABSTRACT FROM AUTHOR]

Multidrug resistance (MDR) mediated by the drug efflux protein, 170-kDa P-glycoprotein (P-gp), is one mechanism that tumor cells use to escape cell death induced by chemotherapeutic drugs. Moreover, evidence suggests that cell lines expressing high levels of 170-kDa P-gp are less sensitive to caspase-mediated apoptosis induced by a wide range of death stimuli, including Fas ligand, tumor necrosis factor, and ultraviolet irradiation. However, the fate of 170-kDa P-gp during apoptosis is unknown. In this study, we demonstrate for the first time that 170-kDa P-gp is cleaved during apoptosis of VBL100 human T-lymphoblastoid CEM cells. Apoptotic cell death was induced by LY294002 (a pharmacological inhibitor of the phosphoinositide 3-kinase/Akt survival pathway), H2O2, and Z-LEHD-FMK (a caspase-9 inhibitor which has been recently reported to induce apoptosis in CEM cells). Using an antibody to a common epitope present in both the third and the sixth extracellular loop of P-gp, two cleavage products were detected, with an apparent molecular weight of 80 and 85 kDa. DEVD-FMK (a caspase-3 inhibitor), but not VEID-CHO (a caspase-6 inhibitor), blocked 170-kDa P-gp cleavage. Recombinant caspase-3 was able to cleave in vitro 170-kDa P-gp yielding two fragments of equal size to those generated in vivo. Considering the size of the cleaved fragments and their reactivity with antibodies, which recognize either the N-half or the C-half region of the protein, it is conceivable that the cleavage occurs intracytoplasmically. Since 170-kDa P-gp has been reported to counteract apoptosis, its cleavage may be a mechanism aimed at blocking an important cell survival component. J. Cell. Physiol. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Recent studies suggest that the prosurvival signal transduction pathway involving phosphoinositide 3-kinase (PI3K)/Akt can confer an aggressive, apoptosis-resistant phenotype to acute leukaemia cells. We have investigated the effect of modulating this signalling pathway on the sensitivity of leukaemic cell lines (NB-4, CEM, Jurkat, MOLT-4) and acute promyelocytic primary blasts to apoptosis induced by 1 μmol/l As2O3. Whereas parental NB-4 cells did not display any phosphorylated (active) Akt, CEM, Jurkat and MOLT-4 cells exhibited high levels of Akt activation. Consistently, treatment of NB-4 cells with pharmacological inhibitors of the PI3K/Akt pathway ( , wortmannin) did not increase sensitivity of these cells to arsenic trioxide (As2O3), whereas siRNA knock-down of Akt enhanced As2O3-induced apoptosis of CEM, Jurkat and MOLT-4 cells. Overexpression of a constitutively active Akt cDNA rendered NB-4 cells less susceptible to As2O3. Upon prolonged exposure to As2O3, we isolated a NB-4 cell clone that was resistant to As2O3 and displayed high levels of active Akt. treatment of acute promyelocytic primary blasts with elevated Akt phosphorylation levels resulted in an increased sensitivity to As2O3. These results may provide a rationale for the development of combined or sequential treatment with PI3K/Akt inhibitors to improve the efficacy of As2O3 on acute leukaemias and also to overcome As2O3 resistance. [ABSTRACT FROM AUTHOR]

Activation of the phosphoinositide 3 kinase (PI3K)/Akt signalling pathway has been linked with resistance to chemotherapeutic drugs, and its downregulation, by means of PI3K inhibitors, lowers resistance to various types of therapy in tumour cell lines. Recently, it has been reported that deguelin, a naturally occurring rotenoid, is a powerful inhibitor of PI3K. We investigated whether or not deguelin could enhance the sensitivity to chemotherapeutic drugs of human U937 leukaemia cells and acute myeloid leukaemia (AML) blasts with an activated PI3K/Akt network. Deguelin (10 nmol/l) induced S phase arrest with interference of progression to G2/M, and at 100 nmol/l significantly increased apoptotic cell death of U937. At 10–100 nmol/l concentrations, deguelin downregulated Akt phosphorylation of leukaemia cells and markedly increased sensitivity of U937 cells to etoposide or cytarabine. A 10 nmol/l concentration of deguelin did not negatively affect the survival rate of human cord blood CD34+ cells, whereas it increased sensitivity of AML blasts to cytarabine. Deguelin was less toxic than wortmannin on erythropoietin- and stem cell factor-induced erythropoiesis from CD34+ progenitor cells. Overall, our results indicate that deguelin might be used in the future for increasing sensitivity to therapeutic treatments of leukaemia cells with an active PI3K/Akt signalling network. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate the possible involvement of the phosphoinositide 3‐kinase (PI3K)/Akt survival pathway in determining resistance to arsenic trioxide (As2O3)‐induced apoptosis. We employed a HL60 cell clone (HL60AR) with a constitutively active PI3K/Akt survival pathway, as well as U937 and K562 cells. In addition, we used parental (PT) HL60 cells overexpressing a constitutively active Akt. Selective pharmacological inhibitors of the PI3K/Akt axis (LY294002, wortmannin) were employed to influence the sensitivity to As2O3. While HL60PT cells were sensitive to 2.5 μM As2O3 and died of apoptosis, HL60AR cells were resistant up to 5 μM As2O3. Treatment with either LY294002 or wortmannin lowered resistance of HL60AR cells to As2O3. Also in U937 and K562 cells, inhibitors of the PI3K/Akt axis caused a decrease in As2O3 resistance. Overexpression of constitutively active Akt in HL60PT cells caused the induction of resistance to 2.5 μM As2O3. Conversely, forced expression of a dominant negative Akt in HL60AR cells resulted in a decrease in As2O3 resistance. Moreover, HL60 cell resistance to 2.5 μM As2O3 could be significantly reduced by incubation with SN50, a peptide inhibitor selective for the NF‐κB transcription factor. Taken together our findings suggest that a constitutive activation of the PI3K/Akt pathway, which is increasingly detected in some types of acute myeloid leukemia, may contribute to As2O3 resistance, most likely through NF‐κB activation. Selective pharmacological inhibitors of this survival pathway, as well as of NF‐κB, might be usefully employed in the future to reverse resistance to this treatment. © 2004 Wiley‐Liss, Inc. [ABSTRACT FROM AUTHOR]

The phosphoinositide 3-kinase/Akt signalling pathway is a recently recognized important parameter in the prognosis and the response to treatment of acute myeloid leukaemia (AML). Akt kinase is activated by phosphorylation on Thr 308 and Ser 473. Active Akt promotes cell growth and survival to apoptotic insults. Thus, it seems important to evaluate Akt phosphorylation in AML blasts. This work aimed to establish whether it was possible to detect Akt phosphorylation on Ser 473 of AML blasts by means of flow cytometry. High levels of Akt activity and phosphorylation were detected in 13 of 15 cases of AML. Flow cytometric analysis revealed similar patterns of Ser 473 expression as was observed with Akt kinase activity and Western blot analysis of Thr 308 and Ser 473 phosphorylation. Double immunostaining enabled the simultaneous flow cytometric detection of an AML-associated antigen (CD33) and Ser 473 phosphorylated Akt in leukaemic blast populations. Our results indicate that flow cytometry enabled the rapid and quantitative assessment of Ser 473 phosphorylated Akt of AML blasts that, when used in combination with cell surface staining, can provide more accurate phenotyping of AML blasts. [ABSTRACT FROM AUTHOR]

Activation of the phosphoinositide 3-kinase (PI3-K)/Akt signalling pathway has been linked with resistance to chemotherapeutic drugs, and its down-regulation, by means of pharmacological inhibitors of PI3-K, considerably lowers resistance to various types of therapy in cell lines derived from solid tumours. Recently, a new class of Akt inhibitors, referred to as phosphatidylinositol ether lipids (PIAs), have been synthesized. We tested whether two new PIAs could lower the sensitivity threshold to chemotherapeutic drugs of human leukaemia cell lines with an activated PI3-K/Akt network. We used HL60AR (for apoptosis resistant), K562 and U937 cells. The two pharmacological inhibitors, used at 5 μmol/l, down-regulated Akt kinase activity and phosphorylation. Neither of the two chemicals affected the activity of other signalling proteins in the Akt pathway, such as phosphoinositide-dependent protein kinase-1 or PTEN. When employed at 5 μmol/l, the Akt inhibitors markedly reduced the resistance of the leukaemic cell lines to etoposide or cytarabine. Remarkably, a 5 μmol/l concentration of the inhibitors did not negatively affect the survival rate of human cord blood CD34+ cells. Overall, our results indicate that new selective Akt pharmacological inhibitors might be used in the future for overcoming Akt-mediated resistance to therapeutic treatments of acute leukaemia cells. [ABSTRACT FROM AUTHOR]

Heparin-induced thrombocytopenia (HIT) is a major complication of heparin therapy. A quick and reliable laboratory assay for the combined determination of pathogenic anti-heparin and platelet factor 4 (H:PF4) antibodies in the serum and platelet activation is not currently available. We developed a new single-tube assay in flow cytometry that combines the detection of antibodies in the serum and their activatory properties on platelets. The assay was tested on 13 serum samples from patients with suspected HIT and six samples from normal donors. The presence of anti-H:PF4 antibody complexes was detected by H:PF4-coated beads, and donor platelet activation induced by HIT sera was determined by Annexin V binding. All data were compared with the patients' clinical setting, laboratory tests, and standard enzyme-linked immunosorbent assay detection of anti-H:PF4 antibodies. This flow cytometry assay allowed unequivocal, simultaneous detection of anti-H:PF4 antibodies in sera and their activatory properties on platelets. All cases for which the diagnosis of HIT was confirmed were detected by the flow assay. This assay, combining for the first time functional and nonfunctional testing on anti-H:PF4 antibodies, is likely to influence the clinical decision for the management of HIT patients. © 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Two distinct types of cell death have been described: apoptosis and necrosis. However, it is becoming increasingly clear that the differences between these two types are far less numerous than initially thought. Morphological analyses might provide important information to distinguish apoptotic from necrotic samples. We recently reported that in necrotic, but not apoptotic, HL-60 human myeloid leukaemia cells, the nuclear protein topoisomerase IIα concentrated in nucleoli. In order to ascertain whether or not this phenomenon was restricted to a peculiar cell type or could be detected also in cells of lymphoid lineage, we performed an investigation aimed at defining the localization of topoisomerase IIα in apoptotic and necrotic Jurkat human T lymphoblastoid cells. Immunofluorescence staining demonstrated that topoisomerase IIα was excluded from the condensed chromatin of apoptotic cells, whereas in necrotic cells it was localized in discrete nuclear dots. Immuno-electron microscopy analysis showed that topoisomerase IIα was undetectable in nucleoli of normal and apoptotic cells, whereas it was present in the nucleolus of necrotic cells irrespectively of the type of inducer used (ethanol, H2O2, HgCl2). Taken together, our findings identify topoisomerase IIα as a potential morphological marker useful to discriminate between apoptotic and necrotic cells. Microsc. Res. Tech. 62:192–200, 2003. © 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Background and Objectives Apoptosis affects white blood cells (WBCs) contained in packed red blood cell (RBC) units. This phenomenon was recently described also in residual WBCs after filtration. The aim of this study was to better characterize the residual WBCs postfiltration by using apoptosis markers and morphology. Materials and Methods Immunofluorescence, flow cytometry and cell-sorting techniques were utilized. Results Residual leucocytes of leucodepleted packed RBC units showed increasing values of apoptotic elements in a time-course experiment. We also demonstrated that these elements are positive for APO 2·7 monoclonal antibody (mAb), poly ADP-ribose polymerase (PARP) cleavage and fluorescein isothiocyanate (FITC)-conjugated N -benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), all of which indicate that programmed death is a feature of this population of cells. Phenotypic analysis with CD45 side-scatter gating demonstrated also that CD15 and CD16 granulocyte-associated antigens are present on a subset of postfiltration leucocytes. Moreover, the expression of human leucocyte antigen (HLA) class I antigens is maintained. Sorting of CD45-positive cells and morphological analysis of these samples confirmed that leucocytes in postfiltration units have morphological characteristics of dying cells. Conclusions Our study extends previous observations regarding the morphology and function of apoptotic cells in leucodepleted blood units, which suggested the presence of apoptotic cells in postfiltration leucocytes. Cleaved PARP, APO 2·7 mAb and positivity for the FITC-conjugated Z-VAD-analogous reagent strongly suggest the activation of programmed death pathways. In addition, the maintained granulocyte-associated and HLA class I antigens might recall an immune response in multitransfused patients. [ABSTRACT FROM AUTHOR]

Summary. Early detection of platelet activation is important for the diagnosis and follow-up of several pathological conditions that primarily or secondarily involve platelets in their pathogenesis. The golden standard assay to detect thrombocyte activation is represented by the release of serotonin, classically performed by demanding methodologies, such as high-performance liquid chromatography, 14 C-labelling and enzyme-linked immunosorbent assay (ELISA). We developed a non-radioactive method, based on individual cells, for the detection of serotonin content in activated and resting platelets by flow cytometry. The assay was standardized on cells activated by Ca2+ ionophore or by sera from patients with heparin-induced thrombocytopenia (HIT). Cells were identified by CD41a surface staining and their serotonin content measured by specific antiserotonin intracytoplasmic staining, while their activation was independently shown by annexin V binding. Cellular degranulation was detected by flow cytometry in all the cases that were also positive by standard ELISA. Moreover, multiparametric flow cytometry analysis revealed that, although virtually all activated cells bind annexin V, serotonin was released only by the platelet subset that downmodulates surface CD41a. [ABSTRACT FROM AUTHOR]

Background and Objectives Packed red blood cells (pRBCs) contain apoptotic white cells. We studied apoptotic cells in pRBCs after filtration and at various time-points during storage. Materials and Methods To maintain the same subset of cells, seven pRBC units were pooled in a single bag and divided equally into seven aliquots. Two series of five experiments were performed: in the first we utilized the Biofil RO1 Max filter, and in the second the Pall BPF4 filter was used. One aliquot was immediately leucodepleted while the others were stored at 4°C and filtered on days 3, 7, 10, 14, 21 and 42 of storage. The postfiltration leucocyte counts and apoptotic evaluations were performed by using the Nageotte chamber and flow cytometry. Results The absolute number of residual leucocytes was always less than 0.5 x 10[sup 6] in each experiment. Nageotte chamber counts showed a greater number of white blood cells than flow cytometry during the 42 days of storage. On day O, the percentage of apoptotic cells in non-leucodepleted pRBCs was 1.1 ±0.4 and 1.2 ±0.4, while in filtered pRBCs it was high from day O, at 53.5 ±16.3 and 52 ±18.5, respectively, with Biofil and Pall filters. On day 10 of storage, apoptotic cells reached a percentage of 42.5 ±15.8 and 41.6 ±18.6 in non-leucodepleted pRBCs, while in filtered units an average value of ≈ 90% was found with both filters. Conclusions The percentage of apoptotic cells was higher in leucodepleted than in non-leucodepleted pRBCs. After filtration, the degree of apoptosis was already high on day O, and reached a mean of ≈ 90% by day 10. The difference in residual WBC counts between the Nageotte chamber and flow cytometry could be related to the presence of a high percentage of apoptotic cells in filtered blood components, and to the method used to distinguish viable from apoptotic cells. [ABSTRACT FROM AUTHOR]

Summary A total of 70 serum samples from heparin-induced thrombocytopenia (HIT II) patients, non-HIT patients and healthy subjects, respectively, were studied for the presence of antiheparin/PF4 antibodies. Two enzyme-linked immunosorbent assay (ELISA) assays were compared with the particle gel immunoassay (PaGIA). Beads of the PaGIA kit were also used to evaluate the feasibility of flow cytometric detection of antiheparin/PF4 antibodies in patient samples. Experiments have shown that all samples found positive by ELISA and PaGIA, were also positive when analysed by flow cytometry by an indirect test using the high-density particles coated with heparin/PF4 complexes and a second step fluorescein isothiocyanate (FITC) antihuman immunoglobulin (Ig)G reagent. The procedure was easy to perform, repetitive and beads were promptly visualized by physical parameters, with a very low background. In conclusion, the results of this study suggest that flow cytometry is a reliable method for the detection of antiheparin/PF4 antibodies. [ABSTRACT FROM AUTHOR]

Immunotoxins specific for the CD80 and CD86 antigens were prepared by linking three type 1 ribosome-inactivating proteins (RIPs), namely bouganin, gelonin and saporin-S6, to the monoclonal antibodies M24 (anti-CD80) and 1G10 (anti-CD86). These immunotoxins showed a specific cytotoxicity for the CD80/CD86-expressing cell lines Raji and L428. The immunotoxins inhibited protein synthesis by target cells with IC50s (concentration causing 50% inhibition) ranging from 0·25 to 192 pmol/l as RIPs. The anti-CD80 immunotoxins appeared 1–2 log more toxic for target cells than the anti-CD86 ones. Immunotoxins containing saporin and bouganin induced apoptosis of target cells. The toxicity for bone marrow haemopoietic progenitors of these conjugates was also evaluated. Bouganin and related immunotoxins at concentrations up to 100 nmol/l did not significantly affect the recovery of committed progenitors or of more primitive cells. The saporin-containing immunotoxins at concentrations ≥ 1 nmol/l showed some toxicity on colony-forming unit cells (CFU-C). The expression of the CD80 and CD86 molecules is prevalently restricted to antigen-presenting cells and is also strong on Hodgkin and Reed–Sternberg cells in Hodgkin's disease. Present results suggest that immunotoxins targeting type 1 ribosome-inactivating proteins to these antigens could be considered and further studied for the therapy of Hodgkin's disease or other CD80/CD86-expressing tumours. [ABSTRACT FROM AUTHOR]

Recombinant human monoclonal antibodies against CD152 have been generated by selecting a synthetic phage scFv library with purified CD152-Ig fusion protein. Sixteen scFv fragments were isolated which specifically react with CD152 by enzyme-linked immunoabsorbent assay (ELISA) and Western blot resulting in their clustering into two groups recognizing different antigenic determinants. One group of scFvs (number3, number13, number40, number44, number47, number51, number57, number80 number83) recognized an epitope on CD152 dimer whereas another group (number15, number18, number31, number35, number54, number72, number81) recognized an epitope on both dimeric and monomeric CD152 molecule suggesting their possible use in understanding the subunit structure of CD152 which is still controversial. Sequencing of the VH genes revealed that all the scFvs belonged to the VH3 gene family but they were different in CDR3 length and composition. It was possible to correlate specific CDR3 sequences with reactivity of the two groups of scFvs. Four scFvs, number3, number40, number81 and number83, each representative of one specific CDR3, were selected for further analysis. Competition ELISA experiments showed that they recognize CD152 in its native configuration and bound to different epitopes from the CD80/CD86 interaction site. The scFvs were able to stain human T lymphocytes stimulated either with anti-CD3 and CD28 antibodies or PHA, PMA and ionomycin by cytofluorimetry suggesting that they can be useful reagents for monitoring the kinetics of surface-bound and intracellular CD152. [ABSTRACT FROM AUTHOR]

Immunotoxins were prepared with three ribosome-inactivating proteins (RIP), momordin, pokeweed antiviral protein from seeds (PAP-S) and saporin-S6, linked to the anti-CD22 monoclonal antibody OM124. These immunotoxins inhibited protein synthesis by CD22-expressing cell lines Daudi, EHM, BJAB, Raji and BM21 with IC50 (concentration causing 50% inhibition) ranging from < 5 × 10-15 to 7.6 × 10-11 M as RIP, and IC90 (concentration causing 90% inhibition) ranging from 5 × 10-14 to 5 × 10-8 M, with no effect on a CD22-negative HL60 cell line at the highest concentration tested (5 × 10-8 M). Apoptosis was induced in sensitive cells. The formation of bone marrow colonies was inhibited by no more than 40% by the immunotoxins at concentrations up to 10-9 M. Treatment with the immunotoxins, alone or in combination, significantly extended the survival time of mice bearing transplanted Daudi cells. A treatment with cyclophosphamide and OM124/saporin immunotoxin was particularly effective in SCID mice transplanted with a low number of cells (3 × 10-6), when 60% of the animals remained tumour-free. [ABSTRACT FROM AUTHOR]

Bromodeoxyuridine (BrdUrd), an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase. In this paper a determination of the S-phase in BLV+ cattle with lymphocytosis has been performed by incorporating bromodeoxyuridine in the DNA. This evaluation was compared to the DNA content, demonstrating that i) bromodeoxyuridine incorporation is a reliable marker of S-phase in BLV+ cattle with lymphocytosis and ii) cytofluorimetry is the method of choice, together with immunocytochemistry, to demonstrate bromodeoxyuridine incorporatioin. Zusammenfassung S-Phasen-Auswertung von Lymphozyten aus mit Bovinem Leukämie-Virus (BLV) infizierten Rindern mittels Brom-Deoxyuridin Das Thymidinanalog Brom-Deoxyuridin wird in die DNS eingebaut und kann daher mit monoklonalen Antikörpe***rn nachgewiesen und als Marker für die S-Phase der Zellteilung eingesetzt werden. In der vorliegenden Arbeit wurde dieser Nachweis an Lymphozyten aus Rindern mit durch BLV hervorgerufener Lymphozytose durchgeführt. Der Einbau von Brom-Deoxyuridin wurde mit dem DNS-Gehalt der Zellen verglichen. Dabei konnte festgestellt werden, daß a) der Einbau von Brom-Deoxyuridin ein zuverlässiger Marker für die S-Phase von Lymphozyten aus BLV-positiven Rindern und b) die Zytofluorimetrie zusammen mit Immunozytochemie für den Nachweis des Brom-Deoxyuridineinbaus die beste Auswertungsmethode ist. [ABSTRACT FROM AUTHOR]