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Biomedical subjects

G Bergamaschi

Publications and source records attributed to G Bergamaschi.

At least 55 records · Page 3Linked to original sources
PubMed1993

Inhibitors of tyrosine phosphorylation induce apoptosis in human leukemic cell lines.

Experimental evidence suggests that hematopoietic growth factors promote cell survival by suppressing apoptosis or programmed cell death. Since interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) induce tyrosine phosphorylation of a common set of proteins in the factor-dependent cell line M07e, we have investigated whether growth-factor-induced tyrosine phosphorylation is involved in the promotion of cell survival and suppression of apoptosis. Experiments were carried out with the leukemic cell lines HL-60 and M07e and the tyrosine kinase inhibitors genistein and tyrphostin AG82. Both the tyrosine kinase inhibitors induced apoptosis of HL-60 and M07e cells. This was indicated by the appearance of DNA degradation and morphologic evidence of nuclear condensation and fragmentation. It was also confirmed by flow cytometry of DNA, which showed apoptotic cells as a fraction of cells characterized by a diminished DNA stainability, represented on the DNA frequency histograms as a distinct peak below the G0/G1 population. Kinase inhibitors also reduced the fraction of cells in the S phase of the cell cycle. That tyrphostin specifically inhibited tyrosine kinases was further suggested by the prevention of its effects by the tyrosine phosphatase inhibitor sodium orthovanadate (vanadate), at least during the first 18-24 h of treatment. The incomplete prevention of genistein effects by vanadate suggests that genistein is a less specific inhibitor of tyrosine kinases than tyrphostin, and may also act as an inhibitor of topoisomerase II. Vanadate also prevented apoptosis and reduction of the S phase in M07e cells cultured for 24 h in the absence of growth factors. These results suggest that tyrosine phosphorylation is an essential step in IL-3 and GM-CSF signal transduction. Since in our experimental model the effects of tyrosine kinase inhibition and growth factor deprivation could be reversed by concomitant inhibition of tyrosine phosphatases, it is suggested that a balance between tyrosine kinases and tyrosine phosphatases establishes whether a cell will survive or undergo apoptosis.

Apoptosis↗
PubMed1993

[Choledochoduodenal anastomosis by laparoscopy].

Eleven patients underwent choledochoduodenostomy under laparoscopic control: 5 for adenocarcinoma of head of pancreas, including 2 with extension into duodenum, 3 for chronic pancreatitis. 1 for gastric carcinoma with pancreatic infiltration 1 for carcinoma of ampulla and 1 for stenosing papillitis. Mean duration of operation was 97.9 minutes and mean hospital stay 7.8 days. No immediate or delayed postoperative complications were reported. The advantages of this method are the marked reduction in recovery time, especially in severely debilitated elderly patients, and the absence of postoperative pain.

Adenocarcinoma↗
PubMed1992

Subcutaneous erythropoietin for treatment of refractory anemia in hematologic disorders. Results of a phase I/II clinical trial.

We have used recombinant human erythropoietin (rHuEPO) in a phase I/II clinical trial to evaluate its ability to reverse refractory anemia in hematologic disorders. rHuEPO was administered subcutaneously 5 days per week at escalating doses (50 to 150 U/kg per day). The aim of treatment was a hemoglobin (Hb) level greater than or equal to 10 g/dL without blood transfusion. Of 25 patients treated, 17 were evaluable, most of them with a regular need for transfusion. Eight of these had lymphoproliferative disorders (three cases of malignant lymphoma and five of monoclonal gammopathy) and were exposed to cytotoxic therapy. The other nine patients had hematopoietic stem cell disorders (four cases of myelodysplastic syndrome, three of idiopathic myelofibrosis, and two of chronic myelogenous leukemia). All patients with lymphoproliferative disorder had serum EPO levels inappropriately low for the degree of anemia, while patients with stem cell disorder showed variable values. Erythroid marrow activity was inadequate in all cases. Seven of eight patients with lymphoproliferative disorder responded to treatment maintaining Hb above 10 g/dL without transfusion. The median dose of rHuEPO required for correction of anemia was 75 U/kg. In four cases response was maintained with 50 U/kg, three times per week. There was no complete response among patients with hematopoietic stem cell disorder, although transfusion requirement was eliminated or reduced in four cases. Four patients developed functional iron deficiency during rHuEPO treatment and required iron supplementation to obtain response. Aggravation of splenomegaly was observed in two cases of myeloproliferative disorder. We conclude that: (1) subcutaneous administration of rHuEPO can be effective and safe in patients with lymphoproliferative disorder exposed to chemotherapy and showing inappropriate EPO response to anemia; (2) this is less likely in hematopoietic stem cell disorders, although favorable responses may be observed in occasional patients; and (3) functional iron deficiency as a cause of nonresponse to rHuEPO is frequent also in nonrenal anemia.

Adult↗
PubMed1992

[Laparoscopic cholecystectomy using argon bistoury].

A total of 92 patients were submitted to laparoscopic cholecystectomy during the period Autumn 1990-Spring 1991. The dissection of the gallbladder from the hepatic bed was performed in 40 patients using the Argon beam coagulator, in 25 using the monopolar electrocoagulator and in 27 using the Holmio laser beam. In average, with the Argon beam coagulator time procedure was respectively 2.7 and 5.4 minutes shorter than monopolar electrocoagulator and Holmio laser. Only one complication (pneumomediastinum) was correlated with the use of the Argon beam coagulator.

Adult↗
PubMed1992

c-abl function in normal and chronic myelogenous leukemia hematopoiesis: in vitro studies with antisense oligomers.

By using antisense oligomers the functional role of the c-abl proto-oncogene in the in vitro growth of bone marrow hematopoietic progenitors from normal subjects and patients with chronic myelogenous leukemia (CML) has been evaluated. Light density bone marrow cells (LDBMs) were depleted of adherent cells, pre-incubated for 15 h with the appropriate oligomer at a concentration of 14 microns, and then plated in methylcellulose for the evaluation of colony formation. Both anti-exon Ia and anti-exon Ib antisense oligomers produced a significant inhibition of normal day 14 CFU-GM growth in vitro (n = 5, 41 +/- 11%, and 36 +/- 7%, respectively; p less than 0.01). In contrast, normal BFU-E growth was not significantly influenced by antisense oligomers (n = 5, 14 +/- 21% and 7 +/- 19%, respectively; p less than 0.05). These findings were confirmed by plating CD34 positive progenitors. When interleukin 3 (IL-3) (100 ng/ml) was added to the culture medium during the preincubation of LDBMCs, the inhibitory effects of antisense oligomers on normal CFU-GM growth were abolished. Seven patients with CML were also studied, all of whom had cytogenetic evidence of 100% clonal hematopoiesis. In five patients in the chronic phase, antisense oligomers were inhibitory on in vitro growth of both day 14 CFU-GM (37 +/- 20% and 37 +/- 15%, p less than 0.05) and BFU-E (45 +/- 15% and 41 +/- 11%, p less than 0.05), and this inhibition was not removed by pre-incubation with IL-3. No significant effect was observed on cluster or colony formation in two patients with CML in accelerated or blastic phase, and on in vitro growth of clonogenic cells from the Ph1-positive K-562 cell line. These findings (i) confirm previous observations showing a lineage specific requirement of c-abl function in normal hematopoiesis, and (ii) suggest that the residual c-abl expression has a role in chronic phase CML hematopoiesis, as its inhibition impairs both myeloid and erythroid colony formation in vitro.

Base Sequence↗
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↗
PubMed1991

A 5'-truncated c-myc gene variant not associated with a risk of cancer.

By analyzing c-myc specific fragments from white blood cell DNAs of 98 gastric cancer patients and 46 control subjects, we observed 6 unexpected patterns due to presence of a variant c-myc gene in addition to the normal gene. Restriction enzyme mapping indicated that the variant c-myc gene was the result of a 5' deletion including the first exon and part of the first intron. The deleted region, non-coding for the functional c-myc protein, contains sequences involved in the regulation of transcription. We therefore analyzed the c-myc mRNAs from a subject carrying the truncated gene and from a subject homozygous for the normal gene in Northern blotting experiments: the mRNAs were indistinguishable, both qualitatively and quantitatively. Family analysis demonstrated that the truncated gene is inherited in a Mendelian fashion. Population studies showed that the allele, both in patients and in control subjects, reaches a polymorphic frequency (2.1% for the whole sample) and that it is not associated with a risk of cancer.

Blotting, Northern↗
PubMed1991

Establishment and characterization of a B-cell line derived from a patient with a myelodysplastic syndrome which expresses myelomonocytic and lymphoid markers.

We describe a novel continuous B-cell line (PV-90) derived from a patient with myelodysplastic syndrome (MDS) and originating from spontaneous infection with the Epstein-Barr virus (EBV). The patient progressed to acute myeloblastic leukaemia (AML) 5 months after clinical onset of MDS. PV-90 is of clonal origin as indicated by the presence of immunoglobulin (Ig) gene rearrangements, monoclonal surface immunoglobulins, and a single DNA restriction fragment corresponding to the EBV genomic termini. PV-90 cells also express a number of myelomonocytic markers, including alpha-naphthyl acetate esterase (ANAE), coagulation factor XIII, and CD68 antigen. Moreover, PV-90 cells constitutively express the c-fms proto-oncogene mRNA as the patient's blast cells did. Whereas a trisomy 11 (+11) was found in the patient's bone marrow cells, PV-90 cells had a normal karyotype initially, but at 4 months showed two different and independent chromosomal abnormalities: 90, XX, -Y, -Y, t(9;16) (q11;p13), and 90, XX, -Y, -Y, t(17;18) (p13;q21), the latter possibly involving the p53 (17,p13) and bcl-2 (18, q21) proto-oncogenes. The early development of these chromosomal aberrations is consistent with a genetic instability of PV-90 cells. Expression of bi-lineage markers and genetic instability may suggest that PV-90 cells originated from transformation of a myelodysplastic progenitor cell capable of both myeloid and B-cell differentiation. The PV-90 cell line might be useful in a number of studies, including the possible role of c-fms in cell differentiation, pathogenetic mechanisms of human preleukaemia and lineage promiscuity in acute leukaemia.

Acute Disease↗
PubMed1991

Tumor necrosis factor alpha modulates the messenger RNA expression of hematopoietic growth factor genes in fresh blast cells from patients with acute myeloblastic leukemia.

Tumor necrosis factor alpha (TNF-alpha) has been previously shown to modulate the expression of hematopoietic growth factor genes in monocytes and other mesenchymal cells. As acute myeloblastic leukemia (AML) blasts can express and produce hematopoietic growth factors, the influence of TNF-alpha on the accumulation of mRNAs for c-myc, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, IL-6 and IL-1 beta was evaluated in fresh blasts from 13 patients with AML. Total cellular RNA was extracted from blast cells cultured for 24 hours with or without TNF-alpha (500 U/ml). The c-myc transcript level was decreased by TNF-alpha treatment in 9/13 cases, and increased in only one case. Among the growth factor genes, the GM-CSF gene was more often and consistently influenced by TNF-alpha, increased levels of its transcript being observed in 6/13 cases following treatment with the cytokine; in no case was there a reduction of GM-CSF mRNA. G-CSF and IL-6 transcripts were more heterogeneously influenced, whereas the IL-3 transcript was never detected in our AML samples. The IL-1 beta message was present in 8/13 untreated and in 13/13 TNF-alpha treated samples. Moreover, in untreated cells, GM-CSF, G-CSF and IL-6 expression was always associated with IL-beta expression. These findings indicate that TNF-alpha can modulate the levels of growth factor transcripts in AML blasts, and raise questions about the effects of TNF-alpha on leukemic hematopoiesis, considering that TNF-alpha, IL-1 and GM-CSF can synergistically stimulate the growth of AML clonogenic cells.

Gene Expression Regulation, Leukemic↗
PubMed1990

Tumor necrosis factor alpha down-regulates c-myc mRNA expression and induces in vitro monocytic differentiation in fresh blast cells from patients with acute myeloblastic leukemia.

We have studied tumor necrosis factor alpha (TNF-alpha) for its capacity to induce differentiation and to modulate c-myc and c-fms protooncogene mRNA expression in fresh blasts from 10 patients with acute myeloblastic leukemia (AML). Bone marrow blast cells were grown in suspension cultures in the presence of 500 U/ml (62 ng/ml) of TNF-alpha for 7 days. Induction of differentiation was assessed by means of morphology, cytochemistry, immunophenotyping (CD11b, CD13, CD14, CD33), and nitroblue tetrazolium reduction. In all cases, exposure of leukemic blasts to TNF-alpha resulted in phenotypic changes consistent with induction of differentiation, although a marked variability in degree and type of response was observed. The majority of cases developed monocytic morphology and showed significant increases (chi 2 test, p less than 0.05) in phagocytic activity and/or expression of ANAE and myelomonocytic differentiation antigens (CD11b, CD14). TNF-alpha reduced c-myc mRNA level over a period of 24 hr in four of six cases studied: the two cases with no down-regulation were the least responsive in terms of myelomonocytic differentiation. These results confirm those obtained with leukemic cell lines, suggesting that TNF-alpha can induce differentiation of fresh AML blasts, mainly toward the monocytic lineage, and that induction of differentiation seems to be closely linked to down-regulation of c-myc mRNA expression over the first 24 hr rather than to attenuation of cellular proliferation per se.

Bone Marrow↗
PubMed1989

[Evaluation of the intestinal absorption of iron orally administered as chondroitin sulfate in normal subjects].

The behaviour of sideremia has been studied in order to assess the intestinal absorption of iron of a new compound, ferric chondroitin sulfate after oral administration in 12 normal volunteers. After administration of 90 mg of iron as ferric chondroitin sulfate, sideremia rose from a basal value of 88 +/- 27 micrograms/dl to a value of 128 +/- 22 micrograms/dl at the third hour. Variance analysis showed that the increases were statistically significant (F = 27.7; p less than 0.00001). In the same subjects, the test was carried out in accordance with a randomised crossover design in two periods after administration of 91 mg of ferritin iron: sideremia rose from a basal value of 92 +/- 27 micrograms/dl to a value at the third hour of 97 +/- 28 micrograms/dl, moderate increases but statistically significant (F = 3.2; P = 0.0354). Variance analysis by repeated measurements showed that increases in sideremia were significantly higher after iron administration as ferric chondroitin sulfate than after administration of ferritin iron (F = 13.18; p = 0.0042). This study documents the good bioavailability of the iron contained in ferric chondroitin sulfate.

Administration, Oral↗
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↗