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The role of circulating microvesicles (MVs) in Myelofibrosis (MF) and Essential Thrombocythemia (ET) is far to be defined. Here we found that 1) circulating megakaryocyte-MVs were reduced in MF and ET while platelet-MVs were increased; 2) the proportion of circulating megakaryocyte- and platelet-MVs was associated with disease severity in MF; 3) ruxolitinib normalized the profile of circulating megakaryocyte- and platelet-MVs in spleen responders MF patients only. Of note, a cut-off value of 19.95% of circulating megakaryocyte-MVs predicts ruxolitinib spleen response. In light of these findings, circulating megakaryocyte/platelet-MVs may have a tissue specific diagnostic and prognostic role in MF.

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 by means 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 them for 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. Copyright © 2016 John Wiley & Sons, Ltd.

With the continuous discovery of new alternative sources containing mesenchymal stem cells (MSCs), regenerative medicine therapies may find tailored applications in the clinics. Although these cells have been demonstrated to express specific mesenchymal markers and are able to differentiate into mesenchymal lineages in ad hoc culture conditions, it is still critical to determine the yield and differentiation potential of these cells in comparative studies under the same standardized culture environment. Moreover, the opportunity to use MSCs from bone marrow (BM) of multiorgan donors for cell banking is of relevant importance. In the attempt to establish the relative potential of alternative MSCs sources, we analyzed and compared the yield and differentiation potential of human MSCs from adipose and BM tissues of cadaveric origins, and from fetal annexes (placenta and umbilical cord) after delivery using standardized isolation and culture protocols. BM contained a significantly higher amount of mononuclear cells (MNCs) compared to the other tissue sources. Nonetheless, a higher cell seeding density was needed for these cells to successfully isolate MSCs. The MNCs populations were highly heterogeneous and expressed variable MSCs markers with a large variation from donor to donor. After MSCs selection through tissue culture plastic adhesion, cells displayed a comparable proliferation capacity with distinct colony morphologies and were positive for a pool of typical MSCs markers. In vitro differentiation assays showed a higher osteogenic differentiation capacity of adipose tissue and BM MSCs, and a higher chondrogenic differentiation capacity of BM MSCs.

The efficiency of in vitro mesenchymal stem cell (MSC) differentiation into the myocardial lineage is generally poor. In order to improve cardiac commitment, bone marrow GFP + MSCs obtained from transgenic rats were cultured with adult wild type rat cardiomyocytes for 5 days in the presence of difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis and cell proliferation. The percentage of GFP + MSCs showing cardiac myofibril proteins (cMLC2, cTnl) was about threefold higher after DFMO addition (3%) relative to the untreated control (1%). Another set of experiments was performed with cardiomyocytes incubated for 1 day in the absence of glucose and serum and under hypoxic conditions (pO 2 < 1%), in order to simulate severe ischemia. The percentage of cardiac committed GFP + MSCs was about 5% when cultured with the hypoxic/starved cardiomyocytes and further increased to 7% after DFMO addition. The contemporary presence of putrescine in DFMO-treated cells markedly blunted differentiation, while the cytostatic mitomycin C was not able to induce cardiac commitment. The involvement of histone acetylation in DFMO-induced differentiation was evidenced by the strong attenuation of cardiac commitment exerted by anacardic acid, an inhibitor of histone acetylase. Moreover, the percentage of acetylated histone H3 significantly increased in bone marrow MSCs obtained from wild type rats and treated with DFMO. These results suggest that polyamine depletion can represent a useful strategy to improve MSC differentiation into the cardiac lineage, especially in the presence of cardiomyocytes damaged by an ischemic environment.

Background Feline nonflea hypersensitivity dermatitis (NFHD) is a frequent cause of over‐grooming, scratching and skin lesions. Multimodal therapy often is necessary. Hypothesis/Objectives To investigate the efficacy of ultramicronized palmitoylethanolamide (PEA‐um) in maintaining methylprednisolone‐induced remission in NFHD cats. Animals Fifty‐seven NFHD cats with nonseasonal pruritus were enrolled originally, of which 25 completed all study requirements to be eligible for analysis. Methods and materials Cats were randomly assigned to PEA‐um (15 mg/kg per os, once daily; n = 29) or placebo (n = 28) while receiving a 28 day tapering methylprednisolone course. Cats responding favourably to methylprednisolone were then administered only PEA‐um (n = 21) or placebo (n = 23) for another eight weeks, followed by a four week long treatment‐free period. Cats were maintained in the study until relapse or study end, whichever came first. Primary outcome was time to relapse. Secondary outcomes were pruritus Visual Analog Scale (pVAS), SCORing Feline Allergic Dermatitis scale (SCORFAD) and owner Global Assessment Score (GAS). Results Mean relapse time was 40.5 days (±7.8 SE) in PEA‐um treated cats (n = 13) and 22.2 days (±3.7 SE) for placebo (n = 12; P = 0.04). On Day 28, the severity of pruritus was lower in the PEA‐um treated cats compared to placebo (P = 0.03). Mean worsening of pruritus at the final study day was lower in the PEA‐um group compared to placebo (P = 0.04), whereas SCORFAD was not different between groups. Mean owner GAS at the final study day was better in the PEA‐um than the placebo‐treated group (P = 0.05). Conclusion and clinical importance Ultramicronized palmitoylethanolamide could represent an effective and safe option to delay relapse in NFHD cats., Background – Feline nonflea hypersensitivity dermatitis (NFHD) is a frequent cause of over‐grooming, scratching and skin lesions. Multimodal therapy often is necessary. Hypothesis/Objectives – To investigate the efficacy of ultramicronized palmitoylethanolamide (PEA‐um) in maintaining methylprednisolone‐induced remission in NFHD cats. Conclusion and clinical importance – Ultramicronized palmitoylethanolamide could represent an effective and safe option to delay relapse in NFHD cats.

CTLA-4 (CD152) is a cell surface receptor that behaves as a negative regulator of the proliferation and the effector function of T cells. We have previously shown that CTLA-4 is also expressed on neoplastic lymphoid and myeloid cells, and it can be targeted to induce apoptosis. In our study, we have extended our analysis and have discovered that surface expression of CTLA-4 is detectable by flow cytometry on 30 of 34 (88%) cell lines derived from a variety of human malignant solid tumors including carcinoma, melanoma, neuroblastoma, rhabdomyosarcoma and osteosarcoma (but not in primary osteoblast-like cultures). However, by reverse transcriptase-PCR, CTLA-4 expression was detected in all cell lines. We have also found, by immunohistochemistry, cytoplasmic and surface expression of CTLA-4 in the tumor cells of all 6 osteosarcoma specimens examined and in the tumour cells of all 5 cases (but only weakly or no positivity at all in neighbouring nontumor cells) of ductal breast carcinomas. Treatment of cells from CTLA-4-expressing tumor lines with recombinant forms of the CTLA-4-ligands CD80 and CD86 induced apoptosis associated with sequential activation of caspase-8 and caspase-3. The level of apoptosis was reduced by soluble CTLA-4 and by anti-CTLA-4 scFvs antibodies. The novel finding that CTLA-4 molecule is expressed and functional on human tumor cells opens up the possibility of antitumor therapeutic intervention based on targeting this molecule.

Background Hemolytic uremic syndrome (HUS) is the main cause of acute renal failure in early childhood. Most cases are due to intestinal infections from Escherichia coli strains (STEC) which produce by Shiga toxin (Stxs). Stx1 and Stx2 produced by STEC in the gut are absorbed into the circulation and, after binding on polymorphonuclear leukocytes (PMNs), are targeted to renal endothelium. The aim of the present work was the development of a method to detect Stxs bound on circulating PMNs and to diagnose STEC infections in patients with HUS. Methods White blood cells isolated after erythrocytic lysis were incubated with anti-Stxs mouse monoclonal antibodies in the presence of human serum to saturate Fc receptors on PMNs. After incubation with fluorescein isothiocyanate–goat anti-mouse immunoglobulin G, flow cytometric analysis was used to demonstrate the cell-bound fluorescence. Results The method was quick (3 h), sensitive (femtomoles), and capable of detecting both Stxs. The presence of Stxs was detected on PMNs from six patients with HUS: four patients had serologic or microbiological evidence of STEC infection, whereas the other two patients had no evidence of STEC infection when employing the standard diagnostic methods. Conclusions The method described is rapid, simple, and based on commercially available reagents, and it might be more sensitive than the standard methods for diagnosis of STEC infection. It also allows the detection of Stxs in blood, a key step to monitor the pathogenesis of HUS. © 2004 Wiley-Liss, Inc.

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 (OrSense) 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 (HemoCue) 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 OrSense 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 OrSense 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.

Recently, electrospray ionization mass spectroscopy (ESI-MS) has been widely used for the identification of drugs of abuse and their metabolites in biological samples. However, the sensitivity and selectivity of this technique are commonly inadequate for the analysis of tetrahydrocannabinol (THC) and its metabolites at very low levels, such as those sometimes required in forensic and clinical-legal applications. We coupled electrospray ionization and surface-activated chemical ionization (ESI-SACI) to various types of mass analyzers (ion trap, triple quadrupole and orbitrap) (ESI-SACI-MS) to improve the detection of 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH), the most common marker of THC abuse. The benefits of this approach in terms of sensitivity and selectivity compared with a common ESI-MS approach are clearly demonstrated. Copyright © 2011 John Wiley & Sons, Ltd.

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

B5-fixed/paraffin-embedded Jamshidi needle biopsies from 125 multiple myeloma patients were reviewed according to both morphological and immunohistological criteria. At microscopic examination, the following parameters were evaluated: i) grade of malignancy (low = 56; intermediate = 50; high = 19); ii) growth pattern (interstitial +/- sheets/nodules = 90; nodular = 13; packed marrow = 18; sarcomatous = 4); III) histological stage (I = 64; II = 35; III = 26). Comparison of the findings in trephine biopsies and aspirates showed that in 30% of the cases the latter led to an underestimation of the tumor burden. Immunohistochemical determination of Ig easily allowed: i) differential diagnosis from exuberant reactive plasmacytosis; ii) recognition and counting of neoplastic plasma cells; iii) detection of minimal residual disease after treatment. Immunohistochemistry also confirmed phenotypic aberration of neoplastic plasma cells, showing positivity for CD45, EMA, and cytokeratins in 14%, 59%, and 25% of the cases, respectively. Furthermore, it displayed expression of the P-glycoprotein in 4/8 resistant cases. These findings underline that routinely processed Jamshidi needle biopsies can be of great value in the study of patients with multiple myeloma.

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

Immunotoxins have been prepared with 3 ribosome-inactivating proteins (RIPs), namely, momordin, pokeweed anti-viral protein from seeds (PAP-S) and saporin, linked to the Ber-H2 monoclonal antibody directed against the CD30 antigen of human lymphocytes. Either the RIPs or the immunotoxins induced apoptosis in the CD30+ L540 cell line, as shown by the morphological aspects of the cells, by the DNA fragmentation visible at the electrophoresis, and by the formation of DNA breaks evidenced by 2 cytofluorometric techniques (propidium-iodide staining and fluoresceine-isothiocyanate conjugate dUTP incorporation). The AC50 (concentration causing apoptosis in 50% of the cells) is in the range 10(-8) to 10(-7) M in the case of RIPs, and 10(-11) to 10(-10) M in the case of the immunotoxins.

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 microM As2O3 and died of apoptosis, HL60AR cells were resistant up to 5 microM 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 microM 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 microM As2O3 could be significantly reduced by incubation with SN50, a peptide inhibitor selective for the NF-kappaB 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-kappaB activation. Selective pharmacological inhibitors of this survival pathway, as well as of NF-kappaB, might be usefully employed in the future to reverse resistance to this treatment.

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.

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 micromol/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 micromol/l, the Akt inhibitors markedly reduced the resistance of the leukaemic cell lines to etoposide or cytarabine. Remarkably, a 5 micromol/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.

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, 14C-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.

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 R01 Max filter, and in the second the Pall BPF4 filter was used. One aliquot was immediately leucodepleted while the others were stored at 4 degrees 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 106 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 0, 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 0, 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 approximately 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 0, and reached a mean of approximately 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.

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 β-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 β-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.

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.

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 (#3, #13, #40, #44, #47, #51, #57, #80 #83) recognized an epitope on CD152 dimer whereas another group (#15, #18, #31, #35, #54, #72, #81) 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, #3, #40, #81 and #83, 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.

BACKGROUND: The aim of the present study was the evaluation of the apoptosis in residual white cells (WBCs) contained in platelet concentrates (PCs) and of the relationship of this apoptosis with the concentration of inflammatory cytokines in the medium and with platelet activation. STUDY DESIGN AND METHODS: Three independent methods were used to evaluated apoptosis in WBCs present in 9 PCs, either from single donors by apheresis (SD-PCs) or from pooled buffy coats (BC-PCs). All PCs were divided in two parts, one of which was irradiated. PCs were stored up to 4 days at room temperature, and samples were withdrawn daily for analysis of apoptosis, of platelet activation (surface and soluble CD62P), and of cytokine concentration (interleukin [IL]-1α , IL-1β, IL-6, IL-8, and tumor necrosis factor α ). RESULTS: Apoptosis was found to occur with storage in both irradiated and nonirradiated units. Platelet activation increased with storage time and was higher in BCPCs. The amount of released cytokines was rather variable among PC units. Only IL-8 was consistently found to increase with storage time. CONCLUSIONS: Apoptosis of residual WBCs occurred in PC units as a function of storage time. The amount and the time course of apoptosis seem to correlate with IL-8 release rather than with platelet activation or with the occurrence of febrile nonhemolytic transfusion reactions.

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

The anti-CD30 immunotoxin (IT) Ber-H2/saporin is effective in patients with refractory Hodgkin's disease. However, responses are short and partial, one of the main reasons being the inability to repeat IT doses because of formation of human antibodies against the murine antibody and/or the toxin. To overcome this problem, we constructed two new anti-CD30 ITs by covalently linking the mouse monoclonal antibody Ber-H2 to the type 1 ribosome-inactivating proteins (RIPs) momordin (MOM) and pokeweed antiviral protein from seeds (PAP-S), which do not cross-react with each other or with saporin. Both ITs inhibited protein synthesis by Hodgkin's disease and anaplastic large-cell lymphoma (ALCL)-derived CD30+ target cell lines with a very high efficiency (IC50 ranging from < 5 x 10(-13) M to 2.75 x 10(-11) M, as RIP). In a SCID mouse model of xenografted CD30+ human ALCL, a 3d treatment with non-toxin doses of Ber-H2/MOM (50%LD50), started 24 h after transplantation, prevented tumour development in about 40% of the animals and significantly delayed tumour growth rate in the others. Main toxicity signs in mice and rabbits were dose-related increase of serum transaminases (AST and ALT) and creatine phosphokinase (CPK). LD50 (as RIP) in Swiss mice was 7 mg/kg for Ber-H2/MOM and 0.45 mg/kg for Ber-H2/PAP-S. Sequential administration of two anti-CD30 ITs (Ber-H2/MOM and Ber-H2/saporin) was well tolerated and did not result in formation of antibodies cross-reacting and with the two plant toxins. The results presented in this paper suggest that in the future, sequential administration of anti-CD30 humanized antibodies linked to antigenically distinct type 1 RIPs (saporin, MOM, PAP-S) should be feasible.

We performed a flow-cytometric analysis of the expression of D antigen in D+W samples (previously termed Du). We also analysed a series of D-positive and D-negative (cde phenotype) samples to obtain positive and negative controls, respectively. The evaluation was carried out by immunofluorescence and the intensity of positivity was expressed as mean channel value (MCV) of fluorescence. Results demonstrated that D+W samples have lower expression (less than 1 log) than D-positive cases (p > 0.001, Student t test), while cde samples show the same MCVs as negative controls. Moreover, it was also possible to set a grading of D antigen expression and to analyze cases difficult to assess by agglutination only.

BACKGROUND The reduction of white cells in blood components before transfusion by filters with at least 3 log10 depletion may prevent adverse transfusion reactions such as HLA alloimmunization, febrile reactions, transmitted infections, and immunomodulation. A new generation of filters with 4 log10 depletion is now available. STUDY DESIGN AND METHODS The aim of this study is to compare the efficiency of white cell reduction by six commercial filters for packed red cells with 3 and 4 log10 depletion (claimed by manufacturers). The analysis of white cell concentration in the white cell-reduced units was performed by flow cytometry and with a Nageotte chamber. RESULTS The last generation of filters (BPF4, RC400, R01 Plus) show mean residual white cell numbers of 0.18 +/- 0.14, 0.26 +/- 0.21, and 0.25 +/- 0.15 x 10(6), respectively, by flow cytometric analysis and 0.05 +/- 0.04, 0.18 +/- 0.15, and 0.38 +/- 0.23 x 10(6), respectively, by Nageotte chamber evaluation. The 3 log10 depletion filters (R01, Leucostop-4LT-mono, R200) have mean residual white cell numbers of 1.41 +/- 0.92, 2.4 +/- 1.99, and 1.05 +/- 0.64 x 10(6), respectively, by flow cytometric analysis and 3.56 +/- 1.7, 1.67 +/- 1.3, and 3.21 +/- 4.1 x 10(6), respectively, by Nageotte chamber evaluation. The data show that the BPF4, RC400, and R01 Plus filters are likely to be more efficient by 1 log10 reduction than the R01, Leucostop-4LT-mono, and R200 filters. CONCLUSION The most recent generation of filters is able to deplete white cells from packed red cells by 4 log10; in particular, with one of the filters, the residual WBC content was less than 0.5 x 10(6) per unit in all experiments, while two other filters reached that level in 9 of 10 experiments.

In this study we show that in vitro cultured human polyclonal NK cell lines and clones express the Ki-1/CD30 Hodgkin-associated antigen, identified by the BER-H2 monoclonal antibody. The percentage of BER-H2+ cells ranged from 19% to 67% in five polyclonal NK cell lines and was 31% and 20% in two NK clones. The intensity of BER-H2 mAb staining on cultured NK cells was remarkably lower than that found on the L540 Hodgkin's lymphoma cell line. Resting PBL populations that had been enriched for NK cells failed to react with the BER-H2 mAb. Western blot analysis performed on cell lysates from a polyclonal NK cell line and from the NK3.3 NK-like cell line revealed that BER-H2-reactive molecules consisted of two major bands of approximately 110 kD and 100 kD. Two bands displaying an identical electrophoretic mobility were also found in lysates of the L540 cell line. The BER-H2 mAb failed to inhibit the nonspecific activated killer activity of cultured NK cells against both K562 and MeWo tumour target cells. In addition, the BER-H2 mAb was unable to trigger the cytolytic activity of NK cells in a redirected killing assay. The observation that cultured human NK cells express the Ki-1/CD30 antigen may be of relevance for the possible lineage assignment of K11/CD30+ lymphoid cell neoplasia with unrearranged TCR genes.

Summary. An immunotoxin containing an anti-CD30 monoclonal antibody (Ber-H2) and saporin, a ribosome-inactivating protein type 1, is described. It specifically inhibits protein synthesis by Hodgkin derived target cell lines with a very high efficiency (IC50 ranging from 5 × 10–12 M to 5.10∼14 M, assaporin), while irrelevant immunotoxins do not. Present results suggest that this immunotoxin could be used for in vivo therapy as well as for ex vivo bone marrow purging in Hodgkin's disease and CD304+ lymphomas.

Toxicity to Raji cells of the xanthine oxidase/hypoxanthine system is related to the formation of single-strand DNA breaks. DNA damage was proportional to the concentration of xanthine oxidase and to the time of exposure. It was prevented by the absence of hypoxanthine, or by the presence of allopurinol, or both superoxide dismutase and catalase. The release of 51Cr from damaged cells was detectable 12h after the inhibition of cloning efficiency and the production of DNA breakage. These data suggest that DNA damage induced by the oxygen products precedes the severe lesion to the cellular membrane.

Two monoclonal antibodies, MRK16 and C219, both directed at the 170 kDa P-glycoprotein multidrug resistance agent, were applied to frozen sections or cytospin preparations from normal human tissues and 60 non-Hodgkin's malignant lymphomas. Adrenal gland, kidney, liver and pancreas were always stained by the reagents, albeit with slightly different patterns. Brain capillaries as well as macrophages and some elements of the bone marrow, peripheral blood, ovarian stroma and colonic, gastric and jejunal mucosa were positive in all examined preparations. There were differences in the staining patterns with the two antibodies. Among the 60 non-Hodgkin's lymphomas, 25 contained a number of positive cells, which ranged from 2% to 100%. No correlation was seen between the expression of P170 and histological type, stage, clinical symptoms or growth fraction. A close relationship was shown between the presence of P170 positive elements and the clinical course of the disease (P less than 0.001).

Urinary albumin excretion (UAE) was determined by radioimmunoassay in two 24 h urine collections from 125 diabetic children and adolescents and from 71 normal children matched for age and sex. Thirteen patients (10.4%) aged greater than 12 years had microalbuminuria, i.e. log transformed UAE levels above the upper normal range (24.5 mg/24 h). UAE values were positively correlated with age, GH secretion, but not with duration of disease, glycosylated hemoglobin, renal size or N-acetyl-beta-glucosaminidase excretion. Diabetic normoalbuminuric children aged 10 years and older had significantly higher UAE than controls and than younger diabetic patients matched for duration of disease. HLA DR3/DR4 heterozygosity frequency was significantly higher (p less than 0.01) in the microalbuminuric group than in the normoalbuminuric. All microalbuminuric subjects (n = 8) with short duration of disease (3.92 +/- 3.43 yr) developed diabetes at puberty. In conclusion, our cross-sectional study suggests: if a number of factors are combined, i.e. HLA DR3/DR4 heterozygosity, onset of disease at puberty and higher GH values, the probability of developing abnormal levels of UAE will increase.

summary. Anti-human platelet antigens (HPA) alloantibodies are seldom involved in febrile nonhaemolytic reactions (FNHTRs). We describe a case in which anti-HPA-5a alloantibodies are related to an FNHTR. We studied the specificity of the alloantibodies by flow cytometry, ELISA and MACE. Typing of donors and the patient was performed by sequence-specific polymerase chain reaction. The alloantibodies were found reactive with HPA-5a antigens. The patient was HPA-5b/b, whereas the donor of the platelet apheresis involved in the FNHTR was HPA-5a/a. Despite the low frequency of anti-HPA-5a antibodies, they might be responsible for FNHTR.