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Biomedical subjects

L Dezza

Publications and source records attributed to L Dezza.

At least 19 recordsLinked to original sources
PubMed1991

Cytotoxic activity of an anti-transferrin receptor immunotoxin on normal and leukemic human hematopoietic progenitors.

The process of cellular iron uptake involves a specific receptor for the plasma carrier transferrin and a pathway of receptor-mediated endocytosis. Transferrin receptor expression is closely related to the rate of cell proliferation, and conjugates between anti-transferrin receptor monoclonal antibodies and toxins have been shown to have potent cytotoxic activity. We have constructed an anti-transferrin receptor immunotoxin by conjugating the anti-transferrin receptor monoclonal antibody B3/25 to a ribosome-inactivating protein, the saporin-6 (SO6), which is derived from the seeds of the plant Saponaria officinalis. The immunotoxin B3/25-SO6 was tested for in vitro cytotoxic activity against the human cell lines K-562 and HL-60 and against normal human bone marrow hematopoietic progenitors and acute myeloid leukemia clonogenic cells. The immunotoxin proved to be an effective inhibitor of K-562 and HL-60 clonogenic cell growth, in vitro colony formation being completely inhibited at immunotoxin concentrations ranging from 10(-7) to 10(-10) M. B3/25-SO6 markedly reduced the recloning efficiency of HL-60 clonogenic cells at 10(-12) M. Exposure of HL-60 cells in suspension culture to 10(-9) M B3/25-SO6 for 48-72 h completely abolished their clonogenic potential. The immunotoxin was also found to be cytotoxic against normal human bone marrow progenitor cells (burst-forming unit-erythroid and colony-forming unit-granulocyte, macrophage) in a dose-dependent manner. However, exposure of normal colony-forming unit-granulocyte, macrophage in suspension culture to 10(-9) M B3/25-SO6 for 72 h resulted in only 50% suppression of their clonogenic potential. Finally, B3/25-SO6 was found to be a potent inhibitor of in vitro growth of acute myeloid leukemia clonogenic cells. The cytotoxic effects of B3/25-SO6 were shown to be specific, since both saporin alone and irrelevant immunotoxins did not have any effect in the cellular systems examined. We conclude that the immunotoxin B3/25-SO6 has dose-related cytotoxic effects on both normal and leukemic human hematopoietic progenitors. Since there are substantial differences between normal and leukemic progenitors with respect to the proportion of cycling cells and the expression of transferrin receptors, B3/25-SO6 or similar immunotoxins may have clinical application in bone marrow-purging procedures.

Antibodies, Monoclonal↗
PubMed1989

Growth of human hematopoietic colonies from patients with myelodysplastic syndromes in response to recombinant human granulocyte-macrophage colony-stimulating factor.

The in vitro effect of recombinant human GM-CSF (rHuGM-CSF) was tested on bone marrow-derived multilineage (CFU-GEMM) as well as megakaryocytic (CFU-Mk), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitors in a group (n = 16) of patients with myelodysplastic syndromes (MDS). Hematopoietic progenitor cell growth was markedly impaired in MDS patients as compared to normal controls (p less than 0.05, at least). Recombinant HuGM-CSF supported the growth of CFU-GEMM, CFU-Mk, and BFU-E at lower, equivalent, or slightly higher frequencies that those found in cultures plated with medium conditioned by peripheral blood leukocytes (PHA-LCM), but it was invariably ineffective in improving growth values. Recombinant HuGM-CSF supported the growth of granulocyte-macrophage colonies in 15 of 16 cases. The overall incidence (mean +/- SEM) of CFU-GM in cultures containing rHuGM-CSF (5 ng/ml) was significantly higher than the one found in cultures stimulated with PHA-LCM (40 +/- 15 vs. 17 +/- 7, p less than 0.05). Upon culture with rHuGM-CSF (5 ng/ml), in 5 of 15 patients de novo colony formation was observed (8 +/- 4) and in 4 of 15 patients CFU-GM growth (129 +/- 33) fell within normal range. Doses of rHuGM-CSF higher than 5 ng/ml did not result in a further increase of MDS-derived colony formation. It is concluded that rHuGM-CSF (a) does not improve the growth of CFU-GEMM, CFU-Mk, and BFU-E; (b) may completely restore the growth of CFU-GM in a subgroup of MDS patients; (c) while ineffective in improving anemia and thrombocytopenia, its in vivo in MDS may correct leukopenia through an effect at the level of granulocyte-macrophage progenitor cell compartment, at least in a subset of highly responsive patients.

Adult↗
PubMed1989

Effects of desferrioxamine on normal and leukemic human hematopoietic cell growth: in vitro and in vivo studies.

The iron chelator desferrioxamine (DFO) has been previously shown to be an S-phase inhibitor of cell proliferation. To investigate its potential as an antileukemic drug, we first studied the effects of DFO on the in vitro growth of normal human hematopoietic progenitors (CFU-GM and BFU-E) and clonogenic cells from human leukemic cell lines. Then we evaluated the effects of DFO on progression of leukemia refractory to conventional therapy in two individuals. Micromolar concentrations of DFO determined a dose-dependent inhibition of normal progenitor growth, with inhibitory dose 50% (ID50) for CFU-GM and BFU-E being 6.7 and 5.5 microM/liter, respectively. Marked inhibitory effects were observed on clonogenic cells from HL-60 (ID50 = 1.4 microM/liter) and U-937 (ID50 = 3.6 microM/liter) human leukemic cell lines grown in semisolid medium. When DFO was given intravenously to a patient with lymphoid blast crisis of chronic myelogenous leukemia, a marked reduction in circulating blast count was observed. On the contrary, no in vivo effect was observed in a patient with acute nonlymphocytic leukemia having transfusional iron overload. We conclude that: (a) DFO is an inhibitor of both normal and leukemic myeloid cell proliferation in vitro; (b) our limited in vivo observations and a previous case study suggest that intravenous administration of DFO to patients with normal to low plasma iron may result in leukemic cytoreduction in vivo.

Anemia, Refractory, with Excess of Blasts↗
PubMed1988

Cell cycle-related proteins: a flow cytofluorometric study in human tumors.

We used 2-parameter flow cytometry (FCM) to investigate the relationship between the cell cycle phases and 3 proteins whose expression is known to increase in proliferating cells: the surface antigen transferrin receptor (Trf-r), the "cyclin" (a proliferating cell nuclear antigen, PCNA), and the nuclear antigen recognized by the monoclonal antibody (MoAb) Ki-67. FITC-labeled antibodies against Trf-r, PCNA, and the Ki-67-reactive antigen, as well as propidium iodide-DNA distribution, were simultaneously measured on human leukemia HL-60 and K562, and breast carcinoma MCF-7 cell lines and on fresh human leukemic and glioblastoma cells. The 70% ethanol fixation for Trf-r and PCNA and the 95% acetone fixation for Ki-67 plus permeabilization (with 0.1% and 1% Triton X100, respectively, for the surface and the nuclear antigens) produced cell suspensions with negligible cell clumping, high-quality DNA profiles, and bright specific immunofluorescent staining. The investigated proteins have different relationships with the proliferative state of the cell. Trf-r is expressed mainly at the transition from G0/G1 to S-phase. PCNA expression is prominent in late G1 and through S-phase and decreases in G2-M. The Ki-67-reactive antigen is widely distributed in G1, S, and G2-M phases. Knowledge regarding the relationships between proliferation-associated antigens and cell cycle phase in normal and neoplastic cells could improve our understanding of the mechanisms underlying growth regulation and neoplastic transformation. Bivariate FCM is an easy method for obtaining these data from large numbers of cells.

Antibodies, Monoclonal↗
PubMed1988

Effects of recombinant human H-subunit and L-subunit ferritins on in vitro growth of human granulocyte-monocyte progenitors.

We have studied the influence of purified recombinant human H-subunit (rHF, acidic) and L-subunit (rLF, basic) ferritins on in vitro colony formation by normal human granulocyte-macrophage progenitor cells (CFU-GM). Whereas rLF had no significant effect, rHF produced significant decrease in colony formation: mean inhibition of CFU-GM was 38% +/- 13% at 10(-8) M and 22% +/- 13% at 10(-9) M. The inhibitory activity of rHF was lost at 10(-10) M, and was inactivated with a monoclonal antibody recognizing the H subunit, but not with a monoclonal antibody recognizing the L subunit. Although H-type isoferritins were found in normal and leukaemic cells, their concentration in peripheral blood plasma and bone marrow plasma from normal subjects and patients with different haematological disorders including acute leukaemia were 10(-11) M or lower, i.e. levels showing no activity in vitro. We conclude that: (i) acidic H-subunit-rich isoferritins have inhibitory effects on in vitro growth of granulocyte-macrophage progenitors; (ii) levels of these isoferritins in peripheral blood and bone marrow plasma are 2-3 orders of magnitude lower than the effective concentrations in vitro, indicating that these molecules do not behave as circulating regulatory or suppressive factors in vivo.

Bone Marrow Cells↗
PubMed1988

Killing of K562 cells with conjugates between human transferrin and a ribosome-inactivating protein (SO-6).

Cellular iron uptake is mediated by binding of transferrin with specific surface receptors and internalization of the Fe-transferrin-receptor complex. This has been examined as a possible pathway for carrying into leukaemic cells a ribosome-inactivating protein (RIP), SO-6, derived from Saponaria officinalis. Purified human differic transferrin was conjugated with SO-6 and a pool of proteins was obtained, with variable numbers of SO-6 molecules linked to a single transferrin molecule. Human erythroleukaemic K562 cells were grown in the presence of human transferrin, SO-6 and human transferrin conjugated with SO-6. The conjugate was found to be internalized via binding with transferrin receptor. Whereas the presence of unconjugated human transferrin and SO-6 in the medium did not significantly influence K562 cell growth, the conjugated proteins displayed an inhibitory activity on cell proliferation. This was maximal after 72 h at a transferrin concentration of 10(-9) M, with about 50% of cells being killed. Bovine transferrin, present in fetal calf serum, did not appear to compete with human diferric transferrin in binding to K562 cells in suspension culture. In a clonogenic assay, colony formation by leukaemic cells was not influenced by free SO-6 or transferrin, whereas the conjugated proteins were markedly inhibitory (about 100% at 10(-9) M). Our findings indicate that SO-6 can be efficiently carried into mammalian cells via the transferrin-transferrin receptor cycle and exert its ribosome inactivating activity. This is in keeping with the existence of an alternative pathway of transferrin endocytosis in addition to the classic acidic endosome pathway. From a practical viewpoint, conjugates between transferrin and SO-6 can be useful tools for studying the expression of transferrin receptors, and deserve also to be investigated for a possible use in cancer therapy.

Humans↗
PubMed1987

Measurement of ferritin-bearing lymphocytes in man. Preliminary studies on the use of monoclonal antibodies specific for the L and H subunits of ferritin.

We used the monoclonal antibodies LFO3 (specific for the L subunit of ferritin) and 2A4 (specific for the H subunit) in an indirect immunofluorescence test for enumerating ferritin-bearing lymphocytes (FBL). In 13 normal subjects, the geometric mean value of FBL was 4% (range 0-13%) with the monoclonal antibody LFO3, and 3% (range 0-8%) with the monoclonal antibody 2A4. Values in 5 subjects with transfusional iron overload and increased plasma L-type ferritin concentration were 5% (4-7%) and 3% (2-4%), respectively, which is similar to those in normal subjects. Thirteen patients with malignant disease had normal to increased values for plasma ferritin; the circulating protein was largely of L-type with undetectable or very low concentrations of H-type ferritin. In the same patients, the percentage of FBL was greater with the monoclonal antibody 2A4 (geometric mean value 8%; range 3-12%) than with the monoclonal antibody LFO3 (geometric mean value 3%; range, 1-7%). It is concluded that acidic and basic isoferritins can be differently expressed on the surface of peripheral blood lymphocytes, and that the monoclonal 2A4 could be particularly useful in the measurement of FBL in patients with malignancy.

Antibodies, Monoclonal↗
PubMed1987

Effects of recombinant alpha and gamma interferons on the in vitro growth of circulating hematopoietic progenitor cells (CFU-GEMM, CFU-Mk, BFU-E, and CFU-GM) from patients with myelofibrosis with myeloid metaplasia.

Myelofibrosis with myeloid metaplasia (MMM) is a chronic myeloproliferative disorder due to clonal expansion of a pluripotent hematopoietic progenitor cell with secondary marrow fibrosis. No definitive treatment has as yet been devised for this condition, which shows a marked variability in clinical course. To evaluate whether excessive hematopoietic progenitor cell proliferation could be controlled by recombinant human interferon alpha (rIFN-alpha) and gamma (rIFN-gamma), we studied the effects of these agents on the in vitro growth of pluripotent and lineage-restricted circulating hematopoietic progenitor cells in 18 patients with MMM. A significant increase in the growth (mean +/- 1 SEM) per milliliter of peripheral blood of CFU-GEMM (594 +/- 253), CFU-Mk (1,033 +/- 410), BFU-E (4,799 +/- 2,020) and CFU-GM (5,438 +/- 2,505) was found in patients as compared with normal controls. Both rIFN-alpha and rIFN-gamma (10 to 10(4) U/mL) produced a significant dose-dependent suppression of CFU-GEMM, CFU-Mk, BFU-E, and CFU-GM growth. Concentrations of rIFN-alpha and rIFN-gamma causing 50% inhibition of colony formation were 37 and 163 U/mL for CFU-GEMM, 16 and 69 U/mL for CFU-Mk, 53 and 146 U/mL for BFU-E, and 36 and 187 U/mL for CFU-GM, respectively. A marked synergistic effect was found between rIFN-alpha and rIFN-gamma: combination of the two agents produced inhibitory effects greater than or equivalent to those of 10- to 100-fold higher concentrations of single agents. These studies (a) confirm that circulating hematopoietic progenitors are markedly increased in MMM, (b) indicate that these presumably abnormal progenitors are normally responsive to rIFNs in vitro, and (c) show that IFNs act in a synergistic manner when used in combination. Because rIFN-gamma can downregulate collagen synthesis in vivo, this lymphokine could be particularly useful in the treatment of patients with MMM.

Antibodies, Monoclonal↗
PubMed1986

Expression of HLA class II (DR, DQ) determinants by normal and chronic myeloid leukemia granulocyte/monocyte progenitors.

It has been suggested that the expression of certain HLA class II antigens stemming from three distinct loci (DR, DP, and DQ) is important not only in the regulation of the immune response but also on the response of hemopoietic precursors to factors inhibiting myelopoiesis. Changes in the expression of DR antigens may be involved in the pathogenesis of altered cell proliferation in chronic myeloid leukemia, since they result in decreased sensitivity of the colony forming units, granulocyte-macrophage to prostaglandin E and acidic isoferritins. In studies using monoclonal antibodies against monomorphic DR or DQ determinants, in a complement-dependent cytotoxic assay, it was found that nearly all normal and chronic myeloid leukemia bone marrow colony forming units, granulocyte-macrophage express DR antigens. The dose response curve was similar for both normal and leukemic precursors. Leukemic peripheral blood precursors were more sensitive than were normal peripheral blood precursors. Normal colony forming units, granulocyte-macrophage did not express DQ antigens, whereas these were expressed in varying quantities by leukemic cells. This study shows that, in the patients we studied, leukemic cells express DR antigens in amounts comparable to normal. In addition, varying amounts of DQ antigens may be observed on leukemic but not on normal progenitors, perhaps as a consequence of an increase in the number of antigens also expressed by normal cells, though in an amount below the detection threshold of cytotoxicity techniques.

Animals↗
PubMed1986

Effect of acidic and basic isoferritins on in vitro growth of human granulocyte-monocyte progenitors.

Acidic isoferritins have been previously found to be highly potent inhibitors of hematopoietic progenitors at concentrations of 10(-16) to 10(-18) mol/L, and it has been suggested that acidic isoferritin inhibitory activity plays a role in the regulation of normal hematopoiesis and also in the pathogenesis of leukemia. To characterize the ferritin species that affect the in vitro growth of human colony-forming unit-granulocyte-macrophage (CFU-GM), we tested different preparations of basic (L-subunit-rich) and acidic (H-subunit-rich) isoferritins. Three preparations of human liver (basic) ferritin did not show any effects on CFU-GM growth at concentrations up to 10(-9) mol/L, irrespective of the degree of glycosylation. Acidic isoferritins were purified both from HeLa cells and human heart. HeLa cell ferritin did not affect in vitro colony formation. One of two preparations of human heart ferritin, containing 5% glycosylated ferritin, showed a mean inhibition of 26% +/- 8% of the control at 10(-9) mol/L (P less than .02), whereas the other preparation, which contained no glycosylated ferritin, did not show any effect of CFU-GM growth. A preparation enriched for glycosylated acidic isoferritins from human heart was found to produce a mean inhibition of 32% +/- 11% of the control at 10(-9) mol/L (P less than .01), whereas another one was ineffective. A significant part of the inhibitory activity was removed by preincubation with the monoclonal antibody 2A4 directed against human heart ferritin. The present findings indicate that basic isoferritins, ie, the predominant ferritin type in human blood, have no effect on the growth of human CFU-GM, and this is in keeping with indirect clinical evidence. Inhibition of colony formation may be obtained by some preparations of acidic isoferritins that are rich in H subunits and bind to concanavalin A. The mechanism(s) responsible for this are not clear, but the effective concentrations are higher than those found in human blood both under normal conditions and in leukemia. At present, the physiologic significance of the observed inhibitory activity is uncertain.

Ferritins↗
PubMed1985

Immunological reactivity of serum ferritin in patients with malignancy.

Serum ferritin has been suggested as a tumor marker in the diagnosis of certain malignancies and for following the activity or dissemination of the malignant process. Since neoplastic tissues generally contain more acidic isoferritins than their normal tissue counterparts, it has also been suggested that the specific assay of such isoferritins in serum may be of particular value in the diagnosis of malignancy. In this work, we have evaluated ferritin concentration in the serum of normal subjects and patients with acute nonlymphocytic leukemia, Hodgkin's disease, breast cancer and lung cancer by simultaneously using three different immunoassays: an immunoradiometric assay based on polyclonal antibodies against human liver (basic, L-subunit rich) ferritin, a radioimmunoassay based on polyclonad antibodies against HeLa cell (acidic, H-subunit rich) ferritin, and an immunoradiometric assay based on the monoclonal antibody 2A4 raised against human heart (acidic, H-subunit rich) ferritin. Most of the patients studied had increased values for liver-type ferritin in the absence of increased iron stores. Binding of serum ferritin to concanavalin A did not prove to be useful in distinguishing a tumor-specific basic isoferritin. The HeLa ferritin assay was found to be less specific than the heart ferritin assay in the detection of acidic isoferritins, and did not provide any advantage over the liver assay in detecting the increased levels of serum ferritin associated with malignant disease. Heart-type ferritin was found in one-fifth of normal sera and 64% of sera from patients with malignancy. Values were very low compared with those for basic ferritin, ranging from less than 0.1 to 17% of total serum ferritin (geometric mean value 1.3%) in patients with malignancy. These findings indicate that at present there is little application for serum ferritin immunoassays based on antibodies to HeLa cell or heart ferritin in the diagnosis or monitoring of malignant disease. This seems to be due to the presence in human serum of biding factors which are responsible for the rapid clearance of acidic isoferritins from the circulation. The serum concentration of basic ferritin, however, can be useful in the diagnosis and management of some malignancies, and it is possible that studies on cell isoferritins can be important in biologic monitoring of neoplastic disorders. It should also be noted that the increased levels of serum ferritin found in patients with malignancy can exert adverse effects on the host immune response and perhaps an inhibitory effect on hematopoiesis.

Antibodies, Monoclonal↗
PubMed1985

Use of a monoclonal antibody against human heart ferritin for evaluating acidic ferritin concentration in human serum.

Immunoassays for acidic ferritins rich in H subunits have shown that these isoferritins are predominant in some cells such as monocytes and red blood cells but have provided conflicting results about their presence in human serum. We have used an immunoradiometric assay based on a monoclonal antibody against human heart ferritin (monoclonal 2A4) for evaluating acidic ferritin concentration in human serum. This assay proved to be highly specific for acidic isoferritins having more than 60% H subunits. Heart-type ferritin was detected in only one fifth of normal sera and sera from patients with iron overload; values were very low compared with those for basic ferritin. Acidic ferritin was found in relatively high concentrations in most patients with iron deficiency anaemia. In other disease states characterized by increased serum concentrations of basic ferritin, acidic ferritin was always less than 21% of the total ferritin. Dialysis in low-ionic-strength buffer showed that both normal and pathological sera had binding factors for human heart ferritin. We conclude that: (i) human serum contains low concentrations of acidic isoferritins which, at variance with basic ferritin, do not appear to be directly related to the amount of storage iron; (ii) the findings of the present study reinforce the opinion that basic and acidic ferritins have different functional behaviours.

Anemia, Hypochromic↗
PubMed1983

Basic and acidic isoferritins in the serum of patients with Hodgkin's disease.

Ferritin concentration has been measured in the serum of patients with Hodgkin's disease (HD) by radioimmunoassays with monospecific antibodies to liver (basic) and HeLa (acidic) ferritin. Elevated levels of serum ferritin with the liver ferritin assay were found only in patients with systemic disease, and were associated with low serum iron. Basic ferritin levels returned promptly to normal when complete remission was achieved. High levels of serum ferritin with the HeLa ferritin assay were found in 94% of all untreated patients. Acidic ferritin concentration was not related to systemic symptoms or alterations of iron metabolism, and returned to within the normal range only 1-2 yr after complete remission. These findings suggest that basic and acidic isoferritins can be distinguished in terms of biological and clinical significance. Basic ferritin is synthesized by the reticuloendothelial cells and the high values found in patients with systemic symptoms are compatible with the non-specific changes known to occur in the reticuloendothelial system during inflammation. In patients with untreated HD an elevated serum concentration of basic ferritin can be considered a marker of systemic symptoms and, therefore, an unfavourable prognostic factor. Acidic ferritin may be derived from abnormal lymphocytes and/or monocytes, including malignant cells, and its serum concentration may be of value in following the course of remission.

Adolescent↗
PubMed1983

Iron loading in congenital dyserythropoietic anaemias and congenital sideroblastic anaemias.

The relationship between body iron status, degree of anaemia, erythroid expansion, age and sex has been studied in eight patients with congenital dyserythropoietic anaemia (CDA) and two patients with congenital sideroblastic anaemia, who had received no or very few blood transfusions and no medicinal iron during the course of their illness. All patients had increased iron stores. Iron load was mild in three women in the reproductive age and severe in two men, in middle age, who had evidence of parenchymal organ dysfunction. Iron loading, as judged by the plasma ferritin concentration, was independent of the degree of anaemia while it was closely related to the patient's age and the degree of increase in the total erythropoietic activity. It is concluded that patients with CDA or congenital sideroblastic anaemia are at high risk of developing haemochromatosis in middle age. Prophylactic phlebotomy or iron chelation therapy should be considered for such patients.

Adolescent↗
PubMed1983

Ferritin in the red cells of normal subjects and patients with iron deficiency and iron overload.

Red cell ferritin was measured in normal subjects and patients with iron deficiency and iron overload by means of radioimmunoassays with antibodies to liver (basic) and heart (acidic) ferritins. In most of the subjects examined, red cells were found to contain greater amounts of heart-type than liver-type ferritin. The basic ferritin content reflected the abnormal body iron status both in iron deficiency and iron overload while the acidic ferritin content was less closely related to the iron status. The two immunologically different red-cell ferritins probably represent distinct ferritin molecules and may have different metabolic functions within haem-synthesizing erythroid cells.

Anemia, Aplastic↗
PubMed1983

Internal distribution of excess iron and sources of serum ferritin in patients with thalassemia.

Liver and spleen iron concentrations, serum ferritin level and binding of S-ferritin to concanavalin A (Con A) were measured in 12 patients with thalassaemia major or intermedia at the time of splenectomy. All these subjects had increased liver iron concentration, most of them had hepatic fibrosis but none of them had histological evidence of chronic hepatitis. No patient had ascorbic acid deficiency. Serum ferritin concentration was increased in all cases, ranging from 266 to 5504 micrograms/l. In all but 2 subjects most of the protein did not bind to Con A, thus behaving as tissue ferritin. There were highly significant correlations between serum ferritin concentration, amount of blood transfused and liver iron concentration. On the average, iron concentration in the liver was about 3 times that in the spleen. The findings obtained suggest that in patients with thalassaemia major or intermedia most of the iron is deposited in parenchymal tissues and most of the S-ferritin derives by leakage from the cytosol of iron-loaded parenchymal cells. S-ferritin is a valid index of liver iron overload in thalassaemic patients without complications such as viral hepatitis and/or ascorbic acid deficiency.

Blood Transfusion↗