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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]

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]

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]

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]

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]

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. [ABSTRACT FROM AUTHOR]

Focuses on the impact of autoimmune thrombocytopenia in patients with malaria. Impact of direct and indirect flow cytometry on platelets and serum; Autoantibodies against platelet glycoproteins IIb-IIIa and Ia-IIa may be present during malaria and could lead to severe thrombocytopenia; Cause of low platelet count in patients with malaria.

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.

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 x 10(-15) to 7.6 x 10(-11) M as RIP, and IC90 (concentration causing 90% inhibition) ranging from 5 x 10(-14) to 5 x 10(-8)M, with no effect on a CD22-negative HL60 cell line at the highest concentration tested (5 x 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 x 10[-6]), when 60% of the animals remained tumour-free.

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.

Lymphocyte activation antigens, such as CD30, represent suitable target molecules for antibody-driven drug delivery in haemopoietic malignancies. A ribosome-inactivating protein (RIP) type 1 of potential interest for mAb targeting is gelonin, which displays a lower toxicity, as compared to other RIPs. In this study, two anti-CD30/antigelonin bispecific monoclonal antibodies (bimAbs), secreted by hybrid hybridomas, were used to deliver this RIP to CD30+ tumour cells. The two bimAbs, termed D4 and A18, were produced using the same anti-CD30 mAb and two anti-gelonin mAbs, directed to unrelated epitopes of the gelonin molecule. These bimAbs enhanced gelonin toxicity (IC50 5 x 10(-8) M, in the absence of mAbs) against the CD30+ L540 Hodgkin's lymphoma cell line in a protein synthesis inhibition assay. Thus, in the presence of 10(-9) M D4 bimAb, protein synthesis was inhibited with an IC50 of 5 x 10(-10) M as gelonin, whereas with A18 bimAb the IC50 was 8 x 10(-11) M. More interestingly, the combined use of the two bimAbs had a synergistic effect, since the IC50 of gelonin reached 6 x 10(-12) M. Among CD30 tumour cell lines, the Hodgkin's lymphoma L428 was also sensitive to gelonin delivered by bimAbs (IC50 6 x 10(-11) M), whereas the COLE Hodgkin's cell line and the T-ALL Jurkat were completely resistant to the toxic effect of gelonin and bimAbs. COLE and Jurkat cells were also resistant to a gelonin/anti-CD30 conventional immunotoxin, whereas they were sensitive to a saporin/anti-CD30 immunotoxin. This suggests that the resistance to gelonin is not related to a lack of internalization through the CD30 molecule but is associated with some property of the RIP.

In this study we have analysed the CD3 and TCR transcript expression in three CD3- large granular lymphocyte (LGL) expansions. These LGL populations show a heterogenous attern of expression for CD2, CD8, CD16, CD56 and CD57 antigens. LGL1 is CD2+CD8-CD16+CD56+CD57+, while LGL2 is CD2-CD8+CD16-CD56-CD57-; LGL3 is similar to LGL1, except for CD8 antigen expression. Functional analysis has revealed a different behaviour of these LGL expansions in a cytotoxicity assay against the NK-sensitive K562 cell line. LGL1 and 3 display a significant NK-like activity, while LGL2 is inefficient against K562 target cells. TCR and CD3 transcript characterization of LGL expansions 1 and 3 showed that they expressed multiple TCR delta transcripts, a non-functional TCR beta transcript, CD3-zeta and -epsilon mRNA, but they lacked CD3 delta transcript and they lacked or expressed at very low levels of CD3 gamma transcript. On the other hand, LGL2 expressed TCR delta, CD3-gamma, -epsilon and -zeta transcripts, while it lacked CD3 delta mRNA. On the basis of these data, LGL1 and 3 seem to be closely related to peripheral blood mature natural killer (NK) cells, whereas LGL2 displays a pattern of TCR and CD3 expression similar to that found in CD1-2-3-4-8-16-thymocytes.

In this study we describe immunotoxins prepared with different CD2 monoclonal antibodies (mAbs) and a ribosome-inactivating protein, saporin. The CD2 immunotoxins were tested on different models. Anti-CD2-saporin conjugates inhibited protein synthesis by a neoplastic CD2+ cell line (SKW-3) and by an interleukin 2 dependent polyclonal CD2+ lymphoid cell culture (T lymphoblasts), with IC50s ranging from 10(-13) M to 10(-11) M (as saporin). Similar results were obtained with proliferation inhibition tests (3H-thymidine incorporation) on phytohaemagglutinin (PHA) driven lymphoid cultures and on mixed lymphocyte culture activated lymphocytes. Moreover a CD2-ricin A chain conjugate was less effective than an analogous immunotoxin containing the same CD2 mAb and saporin in inhibiting lymphocyte proliferation induced by PHA (IC50 approximately 10(-9) M as ricin A chain versus 10(-12) M as saporin). The conjugates were not toxic on bone marrow stem cells. These results suggest that CD2-saporin immunotoxins could represent an effective tool for CD2+ lymphomas or leukaemias, and for T-dependent immune disorders, such as transplanted organ rejection and graft-versus-host disease.

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.

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 x 10(-12) M to 5 x 10(-14) M, as saporin), 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 CD30+ lymphomas.

Low dose arabinosyl cytosine (ARA-C) is effective for treatment of acute non-lymphocytic leukaemia (ANLL). The mechanism of action is not clearly understood and it was suggested that low doses of the drug could induce leukaemic cells to differentiate. We investigated the effect of low dose ARA-C (20 mg/m2/d, divided into two doses s.c. at 12 h intervals, x 20 d) on the cell cycle distribution of leukaemic cells in four cases of ANLL. By comparison, four other cases of ANLL were studied during treatment with standard dose ARA-C (200 mg/m2/d as a continuous i.v. infusion x 7 d). Both treatments induced an accumulation of leukaemic cells in post G1 phases, at a variable extent and rate. During treatment by low dose ARA-C, the mitotic index (MI) fell slowly to zero in two patients who achieved a complete remission (CR), while it fell but recovered during treatment in the patients who did not achieve a CR. The MI fell rapidly to zero in the four cases treated by standard dose, who achieved a CR. These data are consistent with the known cytotoxic activity of ARA-C, via inhibition and slowing of DNA synthesis leading to defective cell proliferation and to cell death.

OKT9 and Ki-67 monoclonal antibodies have recently been proposed as useful tools for evaluating the growth fraction in malignant tumours, with special reference to non-Hodgkin's lymphomas. In particular, while the former is commonly thought to detect a transferrin receptor present on the cytoplasmic membrane of proliferating cells, the latter recognizes a nuclear antigen, which is expressed in G1, S, G2 and M phase of continuously cycling elements. To further verify their reliability, OKT9 and Ki-67 were applied to seven permanent cell lines (four myeloid and three B-lymphoblastoid) and 100 lymphoid tumours (70 non-Hodgkin's and 30 Hodgkin's lymphomas) phenotypically characterized on frozen sections. The results obtained showed that OKT9 and Ki-67 cannot be employed as equivalent means in assessing the growth fraction. In fact, OKT9 is directed to a transferrin receptor which is not only expressed by proliferating cells, but also by some resting elements. On the other hand, Ki-67 provides a nuclear, easily detectable positivity which is restricted to proliferating cells only. Therefore, it seems to represent the only monoclonal which can confidently be employed in the assessment of the growth fraction. Furthermore, the present study underlines that the immunocytochemical analysis of the proliferation rate in tumours gives similar information to the radionucleide uptake assay, while it represents a more sensitive method than the cytofluorimetric evaluation of the DNA content.