Cristina Tassi

A comparison of anti-lymphocyte immunotoxins containing different ribosome-inactivating proteins and antibodies

Immunotoxins were prepared with several single‐chain ribosome‐inactivating proteins (RIPs type 1) and with the A‐chain of ricin linked to the F(ab′)2 fragment of sheep anti‐mouse IgG. The cytotoxic activity of these conjugates was tested on human lymphocytes pretreated with an anti‐CD3 murine MoAb. The immunotoxins inhibited DNA synthesis in phytohaemagglutinin (PHA)‐stimulated lymphocytes with IC50S (concentrations causing 50% inhibition) ranging from 8.9 × 10−13 to 5.7 × 10−11m (immunotoxins containing dianthin 32, saporin, pokeweed antiviral protein from seeds (PAP‐S), bryodin, momordin, momorcochin, and trichokirin), 1 × 10−8m (immunotoxin containing gelonin) and 5 × 10−9m (immunotoxin containing ricin A‐chain). The immunotoxin containing saporin linked to the anti‐mouse IgG F(ab′)2 fragment was also highly toxic to human lymphocytes pretreated with anti‐CD2, ‐CD3, ‐CD5 and ‐CD45 MoAbs, with IC50s × 10−11m. Immunotoxins were prepared also with saporin linked to MoAbs against various CD antigens. The immunotoxin prepared with the anti‐CD3 antibody had the highest specific cytotoxicity to human lymphocytes.

Short- and long-term haematological surveillance of healthy donors of allogeneic peripheral haematopoietic progenitors mobilized with G-CSF: a single institution prospective study

Summary:Healthy allogeneic donors, who were treated with G-CSF and underwent peripheral blood haematopoietic precursor collection at our Institution, were enrolled in a short- and long-term haematological surveillance protocol for a 5–7-year period. To date, 94 donors have been assessed with a mean follow-up of 30 months (4–84); for 30 subjects, the follow-up is ⩾48 months. During G-CSF administration, 23/94 donors showed a significant platelet count decrease from the baseline. Pre-apheresis platelet decrement correlated with the total G-CSF dose administered, baseline platelet level and donor age. Normal platelet counts returned within 4–8 months. PMN and/or lymphocyte lower values were observed in 55/94 donors 2 weeks after G-CSF administration, with mean drops from the baseline of 40 and 36% for PMN and lymphocytes, respectively. The PMN decrease correlated inversely with donor age, as younger donors were more affected than older ones, whereas the lymphocyte decrease correlated directly with the total blood volumes processed in the apheresis courses, in particular for donors subjected to large volume leukaphereses. Long-term observation showed moderate neutrophil reduction (25% count drop from the baseline) in four of the 30 donors observed for four years or more. 14 donors showed persistent, slight lymphocytopenia (mean drop of 13%) until the third year, with recovery in the fourth year of follow-up.

Autologous bone marrow transplantation with immunotoxin‐purged marrow for advanced multiple myeloma

A system to purge the bone marrow of myeloma cells has been developed in our laboratories with the aim of treating with myeloablative radiochemotherapy patients suffering from advanced multiple myeloma. This system is based on the ex vivo incubation of the marrow with an immunotoxin composed of the 8A monoclonal antibody — that recognizes plasma cells and B‐cell precursors — and the ribosome‐inactivating protein momordin. 8 patients have so far been treated. 4 are surviving from 4 to 18 months after ABMT, whereas 4 died after 1 to 6 months, 2 from infections, 1 from relapsing disease and 1 from veno‐occlusive disease. A marked tumour reduction was observed in all evaluable patients; however, none has achieved complete disappearance of the disease. The hæmopoietic reconstitution was significantly delayed in 3 patients. These preliminary results show the feasibility of this approach in advanced MM patients with heavily infiltrated marrow. The place of ABMT in the treatment of MM remains to be determined; the selection of patients with still responding and less advanced disease would probably produce better results.

Antiapoptotic role of p38 mitogen activated protein kinase in Jurkat T cells and normal human T lymphocytes treated with 8-methoxypsoralen and ultraviolet-A radiation.

A combination of 8-methoxypsoralen and ultraviolet-A radiation (320–400 nm) (PUVA) is used for the treatment of T cell-mediated disorders, including chronic graft-versus-host disease, autoimmune disorders, and cutaneous T-cell lymphomas. The mechanisms of action of this therapy, referred to as extracorporeal phototherapy, have not been fully elucidated. PUVA is known to induce apoptosis in T lymphocytes collected by apheresis, however no information is available concerning the underlying signaling pathways which are activated by PUVA. In this study, we found that PUVA treatment of Jurkat cells and human T lymphocytes up-regulates the p38 MAPK pathway but not the p42/44 MAPK or the SAPK/JNK signaling networks. The use of a pharmacological inhibitor selective for the p38 MAPK pathway, SB203580, allowed us to demonstrate that this network exerts an antiapoptotic effect in PUVA-treated Jurkat cells and T lymphocytes from healthy donors. Moreover, the effect of SB203580 was not due to a down-regulation of the Akt survival pathway which was not activated in response to PUVA. These results may suggest that p38 MAPK-dependent signaling is very important for the regulation of survival genes after exposure to PUVA. Since the therapeutic effect of PUVA seems to depend, at least in part, on apoptosis, further studies on the apoptosis signaling networks activated by this treatment might lead to the use of signal transduction modulators in combination with PUVA, to increase the efficacy of this form of therapy.

Apoptosis in leucodepleted packed red blood cells

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.

Antibodies reactive with neutrophils following allogeneic haematopoietic stem cell transplantation

Abstract: Allogeneic haematopoietic stem cell transplants might induce immunological alterations leading to autoimmune‐like syndromes. In particular neutrophil‐associated antigens could represent the target for autoantibodies against neutrophils in patients receiving an allogeneic peripheral stem cell or bone marrow transplantation, giving rise to granulocytopenia. With this aim we studied prospectively 43 allotransplanted patients for the presence of antibodies reacting with neutrophils (ARN), looking for a correlation with a post‐engraftment neutropenia. Our data showed that the direct test for ARN was positive in 30 patients. Interestingly, 7/7 patients who received a T‐cell‐depleted marrow transplant developed ARN. Antibodies with a specific neutrophil‐antigen reactivity were detected in 4 patients, 1 with an anti‐CD16/FcγRIIIb receptor reactivity and 3 with anti‐NA1 reacting patterns, respectively. From a clinical point of view, it was not possible to demonstrate a close and significant relationship between neutropenia and ARN, although patients showing ARN had slightly lower absolute levels of peripheral neutrophils until 6 months after BMT. In conclusion, ARN may be detected in the majority of patients following allogeneic stem cell transplantation; in addition, since ex vivo or in vivo T‐cell‐depletion leads to a higher percentage of patients positive for ARN, it could be hypothesized that “autoimmune‐like” disorders in transplanted patients might be related to a T‐cell derangement due to different numbers and subsets of T lymphocytes.

M-2 protocol for melphalan-resistant and relapsing multiple myeloma

33 patients with advanced refractory multiple myeloma received a combination of vincristine, cyclophosphamide, carmustine, melphalan and steroids (M‐2 protocol). 20 of them had failed prior chemotherapy with alkylating agents and the remaining 13 patients had relapsed after a response to these drugs. An objective tumour cell mass reduction (≥ 50%) was achieved in 17% of the patients (6% of previously nonresponders and 33% of previously relapsing), while 9 additional patients improved (30–50% tumour reduction), for an overall response rate of 47% (39% for previously nonresponders and 58% for previously relapsing). The median duration of response was 7 months. Thrombocytopenia was the most common toxicity encountered in the study (39% of cases). Our findings indicate that M‐2 protocol is an effective salvage treatment for patients who relapse from previous chemotherapy with alkylating agents. In contrast, results in patients who are primarily resistant to these drugs justify the search for different treatment programmes which can produce greater degrees of tumour reduction.

Phase II study of a new alkylating agent (PTT-119) in resistant-relapsed non-Hodgkin's lymphomas

SummaryIn a phase II study we evaluated the effect and toxicity of a new alkylating agent, PTT-119, in 26 patients with non-Hodgkins lymphomas (NHL) resistant to or relapsed alter other chemotherapy. PTT was scheduled by ascalating the dose from 2.0 to 3.3 mg/kg every 3 weeks. Among 21 evaluable patients with NHL, 12 (57%) showed a good response (CR+PR) to PTT-119. Tolerance was acceptably good; no major side effects related to liver, cardiac, or renal toxicity were recorded. The most commonly recorded side effects were nausea and vomiting, alopecia, and phlebitis; diarrhea and drug-related fever were rarely seen. This report indicates a potential usefulness for PTT-119, a non-cross-resistant alkylating agent, in the treatment of NHL.

Comparison of the DNA Content, Bromodeoxyuridine Incorporation and Ki-67 Antigen Expression in Human Acute Myeloid Leukemia.

The cell kinetics of twenty-two acute myeloid leukemias (AML) were investigated by means of flow cytometry evaluating the S-phase DNA content, bromodeoxyuridine labelling index (BrdUrd L.I.) and Ki-67 antigen expression. Eight patients showed a good correlation between the DNA content and BrdUrd L.I., while nine gave rise to divergent results. In the remaining five patients the S-phase DNA content could not be evaluated due to the presence of an additional aneuploid population. The Ki-67 antigen expression defined the extent of the growth fraction in all cases and allowed for better characterization of the cell cycle. These results suggest that the three methods explore only partly overlapping events; thus, it seems that a reliable picture of the cell kinetics in leukemic populations can only be achieved by combining all these methods.

In situ staining of bromodeoxyuridine positive cells in normal and neoplastic colony-forming units grown on plasma clots

Bromodeoxyuridine, an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase of the cell cycle. In this paper a method for the detection of the labeling index of normal and neoplastic colony-forming units (CFU) growing in plasma clot semisolid medium is described and preliminary results on the cell cycle of 7th and 14th CFU granulocyte-macrophage are discussed.