The importance of erythroid expansion in determining the extent of apoptosis in erythroid precursors in patients with beta-thalassemia major.

Beta-thalassemia major is characterized by ineffective erythropoiesis leading to severe anemia and extensive erythroid expansion. The ineffective erythropoiesis is in part due to accelerated apoptosis of the thalassemic erythroid precursors; however, the extent of apoptosis is surprisingly variable. To understand this variability as well as the fact that some patients undergoing allogeneic marrow transplantation are resistant to the myeloablative program, we attempted more quantitative analyses. Two groups of patients totaling 44 were studied, along with 25 healthy controls, and 7 patients with hemolysis and/or ineffective erythropoeisis. By 2 flow cytometric methods, thalassemic erythroid precursors underwent apoptosis at a rate that was 3 to 4 times normal. Because thalassemic marrow has between 5- to 6-fold more erythroid precursors than healthy marrow, this translated into an absolute increase in erythroid precursor apoptosis of about 15-fold above our healthy controls. In searching for the causes of the variability in thalassemic erythroid precursor apoptosis, we discovered tight direct correlations between the relative and absolute extent of apoptosis and the extent of erythroid expansion as measured either by the absolute number of marrow erythroid precursors or by serum soluble transferrin receptor levels. These results could mean that the most extreme rates of erythroid proliferation lend themselves to cellular errors that turn on apoptotic programs. Alternatively, extreme rates of erythroid hyperplasia and apoptosis might be characteristic of more severely affected patients. Lastly, extreme erythroid hyperplasia could generate such numbers of apoptotic erythroid precursors that marrow macrophages are overwhelmed, leaving more apoptotic cells in the sample.

Increased serum levels of circulating intercellular adhesion molecule 1 predict the risk of graft rejection after bone marrow transplantation for thalassemia.

Beta thalassemia is a hereditary anemia curable by bone marrow transplantation (BMT). Class 3 patients have a much worse outcome with a high incidence of rejection after BMT. Adhesion molecules, including the intercellular adhesion molecule 1 are thought to play an essential role in the rejection process. To investigate whether increased levels of soluble intercellular adhesion molecule 1 (sICAM-1) may be predictive of graft rejection, the pretransplant serum concentration of sICAM-1 of 27 beta thalassemic patients who rejected the graft was compared to that of 68 beta thalassemic patients who achieved a sustained engraftment. Fifty serum samples, obtained from marrow donors, matched for age and sex, served as controls. Beta thalassemic patients had significantly higher sICAM-1 concentrations as compared to controls (P = 0.0001). Significantly increased levels of sICAM-1 were found in the patients who subsequently rejected the graft (mean (95% CI) = 490 ng/ml (440; 540)) as compared to those with sustained engraftment (400 ng/ml (384; 415)), (P = 0.004). The mean level of sICAM-1 in patients with early rejection was significantly higher than that in patients with late rejection (P = 0.04). This may indicate a transfusion-mediated role of sICAM-1: some beta thalassemic patients with high sICAM-1 levels, induced by the transfusion support, may remain immunologically active, despite the conditioning regimen, therefore such patients are likely to have an early graft rejection. Our findings indicate that sICAM-1 could be a useful indicator of immune activation in polytransfused patients with beta thalassemia who have a high risk of rejection. Determination of sICAM-1 has potential clinical implications in predicting which patients may reject after BMT.

Correlation between soluble transferrin receptor and serum ferritin levels following bone marrow transplantation for thalassemia.

This study analyzes the serum transferrin receptor (sTfR) levels in a series of 230 ex-thalassemics with a follow-up of 1 to 9 years after bone marrow transplantation (BMT) for homozygous beta thalassemia. Ex-thalassemics are individuals, cured of homozygous beta thalassemia by BMT, who maintain different degrees of iron overload acquired during the pretransplant period. Both in experimental and clinical conditions, sTfR concentrations have been shown to be a quantitative measure of body iron status. This study was carried out to assess whether the level of sTfR may be of help in determining the extent of iron overload in ex-thalassemics. Patients who received the marrow from their HLA-identical sibling donor heterozygous for beta thalassemia, namely heterozygous ex-thalassemics, displayed significantly higher levels of sTfR than patients transplanted from their normal sibling donors (normal ex-thalassemics). This finding suggests that increased erythropoiesis, albeit in part ineffective in heterozygous ex-thalassemics, is responsible for the sTfR increment. Both heterozygous and normal ex-thalassemics had significant lower sTfR levels than their heterozygous (p < 0.003) or normal (p < 0.0001) donors, respectively. These differences may be ascribed to the presence of iron overload in ex-thalassemics in comparison to their normal or heterozygous donors who did not present excess of iron in the body. A significant inverse correlation between sTfR and serum ferritin levels (r = -0.54, p < 0.0001) was found when normal ex-thalassemics were considered. In heterozygous ex-thalassemics, the lack of correlation between these two parameters may be explained by the enhanced erythropoietic activity of individuals with thalassemic trait. These results suggest that the level of sTfR may be a useful indicator of iron overload in normal ex-thalassemics.

Soluble transferrin receptor following bone marrow transplantation from donors heterozygous for beta thalassemia.

This study analyzes the serum transferrin receptor (sTfR) levels in a series of 184 ex-thalassemic patients with a follow-up of 1 to 9 years after bone marrow transplantation (BMT) for homozygous beta thalassemia. A significant inverse correlation between sTfR and Hb levels was observed (r = -0.36, p < 0.001). Patients who received the marrow from an HLA-identical sibling donor heterozygous for beta thalassemia displayed significantly higher levels of sTfR than patients transplanted from a normal sibling donor (p < 0.001). A cut-off value of 2600 ng/mL of sTfR was established. Only 3 out of 56 (5%) patients who received the marrow from a normal sibling, reached a sTfR value above the cut-off level, while 64 out of 128 (50%) patients transplanted from a heterozygous sibling donor showed sTfR values > 2600 ng/mL (p < 0.001). These results suggest that the level of sTfR helps to identify ex-thalassemic patients with enhanced or normal erythropoietic activity among those transplanted from HLA-identical sibling donors heterozygous for beta thalassemia. The physiologic and clinical significance of different patterns of sTfR levels in ex-thalassemic patients with beta thalassemia trait deserves to be investigated.

Initial characterization of an anti-CD5 monoclonal antibody obtained from leukemic cells infiltrating lymphocytes.

BACKGROUND AND METHODS: CD5 is a monomeric glycoprotein expressed on normal and malignant T cells and on chronic lymphocytic leukemia cells. Murine anti-CD5 monoclonal antibodies (MAbs) were obtained using T cell lines, thymocytes, and blast cells from acute T lymphoblastic leukemia as immunogen. In the present report we describe an anti-CD5 MAb obtained from hybridization of mouse spleen lymphocytes immunized with blast cells from a patient with plasmocytoma who underwent leukemic transformation. RESULTS AND CONCLUSIONS: We demonstrate that this MAb arises from the CD5 determinant of lymphocytes disseminated among the leukemic cells. We present initial characterization of this MAb, which may represent a new CD5 epitope. Potentially, due to its efficient internalization through the cell membrane, this MAb might be used to deliver toxins to immunocompetent T lymphocytes for prevention of acute graft-versus-host disease (aGVHD) after bone marrow transplantation.

p185 HER2/neu epitope mapping with murine monoclonal antibodies.

In order to obtain further information on the biological role of the HER2/neu oncoprotein, 7 new monoclonal antibodies (MAbs) were produced against the p185HER2 extracellular domain. These MAbs, together with two others previously produced, were used to investigate the p185HER2 expression in breast carcinomas and compare the recognized antigenic determinants. The 7 reagents (MGR4,5,6,7,8,9 and 10), were shown to define five distinct epitopes. Three of these MAbs (MGR5,7,10), as well as one previously produced (MGR2), recognize the same epitope (Epitope-1) which seems, therefore, to be highly immunogenic for the murine immune system. Epitope-2 recognized by the MGR4 MAb, appears to be closely related to epitope-1 due to a cross inhibition between MGR4 and MGR10, but not MGR2. Epitope-2 is the only one of the 5 also present on the product of the neu oncogene, the rat analogue of the human HER2/neu gene. None of the reagents against epitope-1 and epitope-2 were found to mediate receptor internalization, whereas MGR6 as well as a previously produced MAb (MGR3), both of which define epitope-3 and MGR8 which defines epitope-4, were found to do so. Epitope-4 like the neu-specific peptide recognized by the reference c-neu Ab3 MAb, was detectable on all p185HER2 positive breast cancer, independently from the quantitative content of the oncoprotein, at variance with the other 4 epitopes whose availability on p185HER2 for the relevant MAbs varied with the degree of overexpression. Epitope-5, recognized by the MGR9 MAb, on the contrary to the other epitopes, was prevalently localized at the basal membrane level of the tumor nodule.

Study of a soluble tumor-associated marker composed of CEA related molecules recognized by three monoclonal antibodies.

Three MAbs, MLuC2, MLuC8 and MLuC9, directed against a molecule that is produced and secreted by carcinoma cells were studied with the aim of developing a double-determinant immunoradiometric assay (DDIRMA). We demonstrated by means of immunoblotting, immunodepletion and DDIRMA techniques, that MLuC9 reacted against the CEA molecule, whereas MLuC2 and MLuC8 reacted against a 90 Kd molecule related to CEA. The DDIRMA performed with the anti-CEA as a catcher MAb and the anti-90 Kd as a tracer MAb was found to be positive with the HT29 soluble extract, which suggests the existence of CEA/90 Kd dimeric molecules. The same reactivity was found when sera from patients with lung carcinomas were tested, which excludes that this molecule could be an artefact due to the cell solubilization procedures. The association between CEA and the 90 Kd molecule was further confirmed by immunodepletion experiments in which the immunoprecipitation with one MAb not only removed the recognized molecule, but also partially immunodepleted the material from the other.

Molecular analysis of HLA class II antigens in bone marrow transplanted thalassemic patients.

BACKGROUND: In this work we investigated whether serologically HLA class II compatible donor-recipient pairs showed differences in restriction fragment length patterns, and whether there is a correlation between the genomic differences observed and the incidence of rejection and acute graft versus host disease (GVHD). METHODS: High molecular weight DNA was extracted from thirty-three transplanted thalassemic patients and from their genotypically HLA identical donors. The DNA was digested with TaqI and PstI restriction enzymes, separated by horizontal electrophoresis and transferred onto nylon filters. RESULTS: Differences at the molecular level were observed in only one patient, who rejected the transplant respect to his donor when DNA was digested with the TaqI restriction enzyme and hybridized with DPB cDNA probe. CONCLUSIONS: Although the molecular analysis revealed a difference between a patient who rejected the transplant and his donor, the RFLP typing confirmed the serological identity of the HLA class II antigens in all the other donor-recipient pairs studied.

Selection of monoclonal antibodies which induce internalization and phosphorylation of p185HER2 and growth inhibition of cells with HER2/NEU gene amplification.

In order to obtain further information on the biological role of the HER2/neu oncoprotein monoclonal antibodies (MAbs) were produced against the p185 extracellular domain. To immunize the mice and screen the hybridoma supernatants we selected a lung adenocarcinoma cell line (Calu-3), which demonstrated an over-expression of p185HER2 measured as the reactivity with polyclonal rabbit serum to the 14-amino-acid carboxy-terminal-HER2/neu. Two MAbs, designated MGR2 (IgG1) and MGR3 (IgG2), selected for reactivity on Calu-3 and negativity on A43I live cells, the reference target cell for EGF receptor expression, were found to immunoprecipitate a 185-kDa molecule. Immunodepletion experiments with the polyclonal antiserum and cross-competition experiments indicated that the 2 reagents recognized 2 different epitopes located on the p185HER2 molecule. One of the 2 MAbs, MGR3, was found to internalize, induce p185HER2 phosphorylation and inhibit tumor cell growth in vitro. These results indicate that MGR3 is directed against a determinant located in the p185HER2 ligand binding site and may compete with the p185HER2 ligand, but is incapable of inducing a complete mitotic signal.

Characterization of a monoclonal antibody directed against the epidermal growth factor receptor binding site.

In this work a new monoclonal antibody (mAb), designated MGR1, which recognizes the epidermal growth factor receptor (EGF-R) binding site, is described. The main characteristic of this mAb is its ability to discriminate between cells that express normal levels of EGF-R from cells with overexpression, the detectability threshold by immunocytochemical tests being 5 x 10(4) receptors/cell of 10 microns diameter. MGR1 was found to inhibit EGF binding on the relevant target cells, and vice versa its binding was inhibited by EGF, which indicated that MGR1 recognizes the EGF receptor binding site. MGR1 exerted an inhibitory effect on both the in vitro and in vivo growth of cells with EGF-R overexpression, but had no effect on cells with a normal expression of the receptor. Tumour growth inhibition in athymic mice was also obtained on already implanted tumours. MGR1 therefore seems to be an adequate reagent for the development of immunotherapeutical approaches suitable for the treatment of tumours with EGF-R overexpression.

Production of monoclonal antibodies against a new carcinoma-associated marker in view of developing a serological test.

By immunizing a mouse with human metastatic breast tumor cells from patient effusions and infiltrated lymph nodes, a monoclonal antibody (MLuC2), which identifies a new carcinoma-associated marker, was raised. The reactivity of this reagent was studied by immunohistochemistry on live and fixed cells from tumor cell lines and on frozen sections from surgical specimens. Besides reacting with 73% of breast carcinomas, MLuC2 also reacted with 93% of non-small cell lung carcinoma (NSCLC) and with a few normal tissues. The MLuC2-recognized molecule (CaMLuC2), whose MW was 90 KDa according to immunoblotting experiments, was found to be detectable in the serum and could therefore be of particular interest for serological diagnostic applications. Since the CaMLuC2 epitope was not polyexpressed on the bearing molecule, we produced a new generation of MAbs in order to define epitopes coexpressed with CaMLuC2 on the same 90 kDa molecule, and which are therefore suitable to develop a double-determinant immunoradiometric assay (DDIRMA) for the detection of this marker in the sera of lung carcinoma patients. Different analyses by immunohistochemistry, binding inhibition tests and DDIRMA, proved that the two new reagents developed, MLuC8 and MLuC9, recognize the same or closely related epitopes, which are however different from CaMLuC2, but which are all present on the same molecule. Preliminary immunoradiometric tests performed on sera from lung cancer and control patients showed a good specificity but a low sensitivity. In fact, only 42% of the 28 tested sera samples from NSCLC patients scored positive despite the fact that more than 90% of the NSCLC expressed the relevant antigen.

Immunologic recovery in thalassemic marrow graft recipients following high-dose busulfan and cyclophosphamide.

T lymphocyte subsets, mitogenic response, and immunoglobulin levels were studied in peripheral blood from 95 thalassemic patients before and at different times after bone marrow transplantation. With the exception of patients receiving more than 100 transfusion units before transplant, who showed an increased percentage of CD8-positive cells, thalassemic patients were essentially immunologically normal. Depressed lymphocyte proliferative response to phytohemagglutinin, concanavalin-A, and pokeweed mitogen; decreased IgG, IgM and IgA levels; and abnormal T subpopulation distribution were observed early after transplant. Long-term transplanted patients showed complete recovery of the immunological profile with the exception of the IgA levels, which were significantly decreased up to 2 years after transplant.

Fetal liver transplantation in the mini-pig.

After the characterisation of the hematological and immunological status of the mini-pig fetus at different gestational ages of development was performed, two different groups of animals receiving 800 rads of TBI given by a radioactive cobalt source at a dose/rate of 5/6 rad/min or 750 rads of TBI given by a Linear Accelerator at a dose/rate of 25/26 rad/min, both in a single dose exposure, were transplanted with a pool of allogeneic fetal liver cells whose age ranged between 55 and 75 days of gestation. In the first group 1 animal out of 6 is alive and well 30 months post-transplant. In the second group one of the nine transplanted animals survived 78 days. Engraftment was proved by the presence of the donor chromosome in the proliferating bone marrow cells in one animal.