Biomedical subjects

G Mazzini

Publications and source records attributed to G Mazzini.

At least 55 records · Page 3Linked to original sources

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 kinetics in human malignancies studied with in vivo administration of bromodeoxyuridine and flow cytometry.

Bromodeoxyuridine (BrdUrd) is a pyrimidine analogue which is incorporated into the DNA of proliferating cells. When in vivo BrdUrd infusion is coupled with bivariate flow cytometry to measure cell BrdUrd incorporation and DNA content, both the percentage of DNA-synthesizing cells [BrdUrd-labeling index (LI)] and the DNA synthesis time (TS) can be determined on the same tissue sample. From experimentally determined LI and TS, the potential doubling time of the population and its cell production rate are calculated. To ascertain whether the BrdUrd infusion method is clinically feasible and if data are reliable, we studied patients with leukemia, refractory anemia, multiple myeloma, and brain and gastric tumors. The BrdUrd incorporation data were compared with those determined on duplicate samples with the techniques conventionally used for LI and TS values, i.e., 3H- and 14C-labeled thymidine autoradiography, respectively. The complete BrdUrd procedure takes 6-9 h, and no immediate toxicity from BrdUrd administration has been observed. In an 8-month period, 154 patients were studied. Successful LI and TS determinations were obtained in 78.9 and 59.7% of cases, respectively, more often in hematological than in solid tumors. The values for LI and TS assessed with the BrdUrd technique were very close to those found with 3H- and 14C-labeled thymidine autoradiography (r = 0.88, P less than 0.005, and r = 0.89; P less than 0.005, respectively). The potential doubling time and production rate were accordingly similar. These data indicate that in vivo BrdUrd infusion coupled with flow cytometry measurements can be performed in clinical settings and that this method is reliable. It could be used for kinetic studies in clinical trials aimed at evaluating the prognostic relevance of proliferative parameters and for planning radio- and/or chemotherapy.

Autoradiography↗

PubMed1988

Age-related changes in the proliferative kinetics of phytohemagglutinin-stimulated lymphocytes. Analysis by uptake of tritiated precursors of DNA, RNA and proteins, and by flow cytometry.

We have examined the age-related changes in the kinetics of lymphocyte proliferative responses to phytohemagglutinin by flow cytometry and by the uptake of tritiated thymidine, uridine and leucine. The lymphocyte suspensions used in these experiments were obtained by umbilical cord blood samples of full-term normal newborns and from the peripheral blood of young (under 30-year-old) or aged (more than 70-year-old) healthy donors. The results indicate that the cord blood lymphocytes were activated by phytohemagglutinin to incorporate tritiated precursors of proteins, RNA and DNA at a more rapid rate than the lymphocytes from young or old donors in the first 3 days of in vitro culture. Flow cytometry confirmed higher percentages of activated cycling cells in umbilical cord blood after 24 h of culture. The lymphocytes from old donors incorporated significantly lower amounts of tritiated precursors of DNA and RNA than lymphocytes from young donors at the third day of culture. The uptake of tritiated leucine by lymphocytes from old donors was significantly reduced up to the sixth day of culture. On the contrary, at the eight day of culture, lymphocytes from old donors incorporated significantly higher amounts of labeled DNA, RNA and protein precursors. In agreement with these findings, flow cytometry demonstrated a trend towards higher percentages of cycling cells in the lymphocyte cultures from old donors after 6 and 8 days. These results indicate an age-related decline in the rate of lymphocyte blastogenesis after in vitro PHA stimulation and suggest that after a delayed PHA-induced activation, the lymphocytes from aged donors have a near-normal ability to proliferate.

Adult↗

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

Cell kinetics of human brain tumors: in vivo study with bromodeoxyuridine and flow cytometry.

Bromodeoxyuridine (BUDR) is a thymidine analog which is incorporated into the DNA of proliferating cells. Since the dose of BUDR needed to label cells is not toxic, cell labelling can be accomplished in vivo, by infusing the substance in patients. A monoclonal antibody against BUDR is then used to identify BUDR-labelled cells. The same cell population can also be stained for DNA content with propidium iodide (PI). Using bivariate flow cytometry (FCM) for measurements, both the percentage of BUDR-labelled cells and their total DNA content can be evaluated. This technique allows one to obtain the labelling index (LI) and the DNA synthesis time (TS). The potential doubling time (Tpot) and the fractional turnover rate (FTR) can be mathematically derived, so that a complete picture of tumor growth can be obtained. Our aim was to ascertain whether this method is clinically applicable and whether the kinetic values obtained are reliable. We studied 22 patients with benign and malignant brain tumors, and observed no immediate toxicity from BUDR administration. The BUDRLI obtained ranged from 0.9% to 3.9% (median: 2.0%) in meningiomas and from 3.8% to 7.6% (median: 6.3%) in malignant gliomas (P less than 0.01). The fraction of S-phase cells determined with the BUDR FCM technique was statistically similar to that found by single DNA flow cytometric analysis performed on duplicate samples of both benign and malignant brain tumors. The TS obtained in malignant gliomas ranged from 10.5 to 227 h (median: 12.8). The calculated Tpot ranged from 7.6 to 26.8 days (median: 11.6), and the calculated FTR ranged from 3.7 to 13.1 cells/100 cells/day (median: 8.8). These data suggest that in vivo BUDR infusion coupled with FCM can be performed in clinical settings, and it is reliable and can easily be used for kinetic studies in clinical trials aimed at evaluating the prognostic relevance of proliferative parameters and in planning tumor treatment.

Adult↗

PubMed1988

Flow cytometric analysis of paraffin-embedded material in human gastric cancer.

Flow cytometric DNa analysis was performed on formalin-fixed, paraffin-embedded samples obtained by gastroscopic biopsy from 9 patients with histologically normal gastric mucosa (36 specimens) and by radical gastrectomy from 42 cases of human gastric cancer (120 specimens). Ploidy patterns and the distribution of cells in the different cell cycle phases were estimated, and the results were correlated with the histologic and clinical features. All samples of normal mucosa showed a diploid modal DNA content whereas DNA aneuploidy was encountered in 71.4% of the gastric tumors. The correlation between aneuploidy and histologic malignancy grading was statistically significant: aneuploidy was found in 36.4% of highly differentiated (grade 1 and grade 2) tumors and in 75.0% of poorly differentiated (grade 3) tumors (P less than .05). The percentage of cells in S-phase in normal gastric mucosa (median: 5.0%) was lower than that in the tumors (median: 11.3%) (P less than .05). There was a trend for grade 3 tumors to have higher median values (median: 13.4%) than grade 1 and 2 tumors (median: 9.3%); however, this was not statistically significant. An aneuploid DNA pattern was associated with a poorer prognosis, both in early and in advanced stages of gastric tumors, while proliferative activity did not correlate with postoperative survival.

Adult↗

PubMed1987

Familial hypertrophy of masticatory muscles.

Three familial cases of idiopathic hypertrophy of the masticatory muscles are reported. The muscular enlargement was noted in late childhood or early adult life, was bilateral, insidious, painless and non-progressive. Electrophysiological and laboratory findings were normal in one case. CT examination, performed in two cases, showed homogeneous bilateral enlargement of masseter muscle bulk consistent with true hypertrophy. Two patients also showed congenital malformations. A review of the literature showed that this disorder is very rare.

Adolescent↗

PubMed1987

Influence of cis-dichlorodiamineplatinum on glioma cell morphology and cell cycle kinetics in tissue culture.

C6 glioma cells (CCL 107) were cultured for three days and then treated with cis-dichlorodiamineplatinum (cis-DDP) at doses of 0.2-10 micrograms/ml medium. Changes in DNA synthesis and DNA content, as well as morphology of cells and chromatin distribution, were examined from the first post-treatment day onwards. The number of cells labelled with [3H]thymidine, detected autoradiographically, decreased after treatment with 0.2-10 micrograms/ml by approximately one half on post-treatment day 1 and diminished further by the third day after treatment. The labelled cells were entirely absent only after treatment with 10 micrograms/ml, 7 days post-treatment. Mitoses decreased from 1.4-0.6% by post-treatment day 1 and completely disappeared by day 3 (1 microgram/ml). Feulgen cytophotometry and propidium iodide cytofluorimetry revealed accumulation of cells in the S-phase, especially the latter part (0.5 and 1.0 micrograms/ml, post-treatment day 1) and subsequently also in G2 phase (post-treatment day 3). Incomplete cyto- and karyokinesis in some cycling cells was indicated by an increased number of binucleate cells and nuclei of higher ploidy classes. Labelled cells with intermediate DNA values were, on average, labelled less intensively, as was revealed by simultaneous measurements of DNA content and [3H]thymidine incorporation. Some cells displayed reduction in grain density over heterochromatin clumps. This would be in agreement with the late S-phase block of DNA replication. After post-treatment day 3 the density of cells in cultures was substantially lower.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Ploidy and proliferative activity of human brain tumors. A flow cytofluorometric study.

Ploidy and proliferative characteristics were estimated by flow cytometry of the nuclear DNA content of 92 human brain tumors. Samples were frozen at -20 degrees C immediately after surgery and single cell suspensions were obtained with a mechanical dissociation technique. Propidium iodide was employed for nuclear DNA staining. Human normal brain tissue was used as internal diploid reference standard. 86% of benign tumors had unimodal DNA distribution with a DNA index (DNA I = modal channel of the G0/1 peak of the studied population/modal channel of the G0/1 peak of the normal brain) usually within the diploid or near-diploid range. 14.0% had aneuploidy, with an additional cell peak having a median DNA I of 1.60. Among malignant tumors, these figures were 61.2 and 38.8% (p less than 0.001). The percentage of S phase cells was higher in malignant (median = 3.6) than in benign tumors (median = 2.0, p less than 0.01), without correlation to histological tumor subtype. Flow cytometry appears to be a useful method for evaluating differences in DNA distribution in tumors of the central nervous system.

Adolescent↗

PubMed1987

DNA-protein flow cytometric analysis in non-Hodgkin lymphoma.

A flow cytometric study of DNA and protein contents was performed on cell suspensions obtained from 73 adult patients with non-Hodgkin lymphoma. Bivariate analysis identified a second subpopulation, not revealed by DNA determination, in 25% of the tumors. Protein heterogeneity was more frequently observed in diffuse than in nodular histology according the Rappaport classification and in high-grade than in low-grade malignancy tumors by the Kiel classification and the Working Formulation, but it was not related to ploidy or cell proliferative rate. The presence of an additional subpopulation, detected by protein analysis, defined as monoclonal by DNA analysis, could adversely affect clinical outcome in terms of response to treatment and overall survival.

Cell Division↗

PubMed1987

Ploidy and proliferative activity of human gastric carcinoma: a cytofluorometric study on fresh and on paraffin embedded material.

Flow cytometric DNA analysis was performed on both fresh and on paraffin embedded samples obtained by gastroscopic biopsies in 5 patients with histologically normal gastric mucosa (20 specimens) and by radical gastrectomies in 9 cases of human gastric cancer (36 specimens). Ploidy and the distribution of cells in the different cell cycle phases were estimated. Results from fresh tissue were compared with those from paraffin embedded material. All the samples of normal mucosa showed a diploid modal DNA content. Unimodal DNA distribution was found both in fresh and paraffin embedded material of 3 well differentiated gastric adenocarcinomas, whereas the remaining 6 tumors (1 moderately differentiated, 4 poorly differentiated and 1 undifferentiated adenocarcinomas) showed aneuploidy. The percentage of cells in S phase in normal tissue and in tumors were respectively 9.0% and 11.9% in the fresh material, and 10.0% and 15.0% in the paraffin embedded material. No statistically significant differences were found between fresh and paraffin embedded samples, whereas the proliferative activity was, in both cases, statistically higher in tumors than in normal mucosa (p less than .01 and p less than .001 respectively). The quality of DNA flow cytometry from paraffin embedded material was comparable with that from fresh samples.

Aged↗

PubMed1987

Flow cytometric determination of ploidy and proliferative activity on isolated bone marrow particles and on total bone marrow aspirate.

The nuclear DNA content distribution of peripheral blood (PB) and bone marrow (BM) cells was determined by propidium iodide flow cytometry in 33 patients who underwent BM aspiration for diagnostic purposes. Two types of BM samples were taken during every aspiration procedure: whole BM aspirate, composed of BM particles contaminated by PB cells; isolated BM particles. Proliferative activity was calculated as the percentage of cells with DNA content intermediate between the diploid (2n) and the tetraploid (4n) values (2n-4n%). Ploidy was expressed as the ratio between the modal channel of the G0-G1 peak of the probe and that of an internal reference standard (DNA index, DI). The 2n-4n% was very close to zero in all PB samples. It was significantly greater in BM particles (21.2 +/- 6.6%) than in whole BM aspirate (16.6 +/- 5.5%, p less than .0005), with a close correlation (r2 = 66; p less than .0001) between the two values. Aneuploid stem lines were found in BM but not in PB. The DI of BM stem lines were similar in whole BM aspirate and BM particles, but the percentage of aneuploid cells was usually higher in BM particles. The reduced proliferative activity and the lower percent of aneuploid cells found in whole BM aspirates, with respect to BM particles, can be attributed to the contamination of BM tissue by PB, which had a very low proliferative activity and did not show aneuploidy. BM particles are therefore an easily obtained and reliable sample for routine evaluation of proliferative activity and ploidy of BM cells by DNA flow cytometry.

Aneuploidy↗

PubMed1986

High-degree abnormalities of nuclear DNA distribution in SIg negative acute lymphoblastic leukemia with vacuolated blasts.

Among 65 consecutive patients with untreated adult acute lymphoblastic leukemia (ALL), six had peculiar clinical and cytologic features. Bone marrow and circulating blasts were large and showed prominent vacuolization, with variable degree of granular periodic acid-Schiff positivity in the cytoplasm. All patients had marked spleen and liver enlargement with cholestasis and without either lymph node or central nervous system disease. They achieved complete response to chemotherapy, and their median survival was longer than 12 months. Blast cells were studied for immunologic markers, propidium iodide flow cytofluorometric DNA content, and in vitro tritiated thymidine labeling index (3H-TdR LI). These showed a "null" phenotype in two cases, contained intracytoplasmic immunoglobulins (Cy mu) in two and were positive for CALL and Ia antigens in two others (one of which also contained Cy mu). Surface immunoglobulins (SIg) were found on less than 12% of cells, and T-cell markers were absent. In five cases, one or two stem lines with highly abnormal modal DNA content were found to coexist with diploid blasts. The 3H-TdR LI was high (8.5%-15.5%). Abnormal cell lines, after being cleared by chemotherapy, were found again at relapse. Additional cell lines appeared during the course of the disease in one patient, having different sensitivity to cytostatics. These cases of ALL apparently derive from B-cells not yet expressing SIg and have an exceptionally high incidence of aneuploid cell lines.

Adolescent↗

PubMed1986

Pericentromeric heterochromatin and A-T contents during Robertsonian fusion in the house mouse.

The pericentromeric heterochromatin of meiotic trivalents formed by the Robertsonian (Rb) chromosomes and the two homologous acrocentrics in the house mouse was evaluated by static cytophotometry after selective staining. To reveal pericentromeric heterochromatin specifically, C-banding Giemsa and Hoechst 33258 stains were utilized. Five different Rb chromosomes were investigated and none of them possessed less pericentromeric heterochromatin than the sum of the two homologous acrocentrics. Moreover the total A-T (DAPI) and DNA (PI) content was quantitatively evaluated, by flow cytometry, in G0/G1 nuclei belonging to four different Rb mouse populations, karyotypically characterized by the presence of up to nine Rb chromosomes. Again there were no significant difference, of DAPI and PI content, in the Rb populations nor between any of them and the NMRI/HAN strain with forty acrocentric chromosomes. We conclude that the main consequence of Robertsonian processes (i.e. the rapid variation of the karyotype structure) does not imply detectable quantitative variation in the genome portion involved in the Rb process. We also discuss the possibility that the high rate of Rb exchange in the house mouse could be favoured by the simultaneous effects of undetectable losses of chromosomal material, high repetitiveness of the DNA involved, the presence of the same major type of satellite DNA over each chromosome and the all acrocentric constitution of the karyotype.

Adenine↗

PubMed1986

Adult acute non-lymphoblastic leukaemia: reliability and prognostic significance of pretreatment bone marrow S-phase size determined by flow cytofluorometry.

46 adult patients with non-lymphoblastic acute leukaemia (AnLL) had pretreatment proliferative activity of bone marrow (BM) blasts determined simultaneously with propidium iodide DNA flow cytofluorometry (as the percentage of cells with DNA content intermediate between the diploid and the tetraploid values, 2n-4n cell %) and in vitro tritiated thymidine cytoautoradiography (as the labelling index, LI). They were then treated with the same chemotherapy regimen, including 2-3 courses of sequential vincristine, arabinosylcytosine and adriamycin, for response induction, and monthly courses of different cytostatics for maintenance. Median 2n-4n cell % was 9.9 and median LI was 5.9. A close linear relationship (r = 0.913, p less than 0.001) between the two parameters was found. Patients having a 2n-4n cell % greater than 15.6 responded more often to chemotherapy than patients having a lower percentage (p less than 0.05) but also experienced a shorter duration of first response (p less than 0.05). Overall survival was longer in patients with 2n-4n cell % greater than 15.6 (p less than 0.02). Multiple regression analysis indicates that 2n-4n cell % is a statistically significant (p less than 0.05) independent factor correlating with duration of response in adult AnLL.

Acute Disease↗

PubMed1986

Flow cytometric evaluation of proliferative activity and ploidy in myelodysplastic syndromes and acute leukemias.

Propidium iodide (PI) DNA distribution of bone marrow (BM) cells was studied by flow cytometry (FCM) in 36 patients without hematologic or malignant disease (normal BM) and in 172 patients with anemias (36 pts), myelodysplastic syndromes (MDS) (33 pts) and acute leukemia (AL) at diagnosis (60 pts), remission (24 pts) and relapse (19 pts). White blood cells from normal male subjects were used as an external diploid reference standard (median CV = 3.8). Patients with normal BM, anemias, MDS and acute leukemia at diagnosis had tritiated thymidine labeling index (LI) and most with MDS and AL had also evaluable cytogenetics performed on the same BM sample used for FCM. In normal BM, median aliquot of cells with PI-DNA content intermediate between the diploid and the tetraploid value (2n-4n cells %) was 15.7. The ratio between the fluorescence intensity of the G0/1 peak of normal BM cells and the fluorescence intensity of the G0/1 peak of the reference standard (FI ratio) ranged from 93 to 1.05 (mean +/- 2SD). The 2n-4n cell % was higher than normal in anemias (p less than .001), lower in leukemias (p less than .001) and widely scattered in MDS. A linear correlation was found between 2n-4n cell % and LI, with 2n-4n cell % value higher than LI. The FI ratio was lower than normal in anemias (p less than .05), higher in AL with normal cytogenetics (p less than .02) and broadly scattered in MDS with normal cytogenetics. From our experience, PI-DNA-FCM is a simple and adequate method to evaluate proliferative activity in hematologic diseases. Nevertheless, caution must be taken in attributing small changes in FI ratio to aneuploidy, since they are found in anemias and in MDS and AL with normal cytogenetics, possibly due to differences in PI uptake by different cell types.

Acute Disease↗

PubMed1986

Proliferative characteristics and ploidy of human brain tumors by DNA flow cytometry.

We studied nuclear DNA distribution by flow cytometry in 59 human brain tumors. Samples were frozen at -20 degrees C immediately after surgery and unicellular suspensions were obtained with a mechanical dissociation technique. Nuclear DNA was stained with propidium iodide. Normal human brain tissue was used as a diploid reference standard. In 86.3% of benign tumors an unimodal DNA distribution with a DNA index usually within the diploid range was found. Among malignant tumors, 64% had un unimodal DNA distribution with diploid or near-diploid modal DNA content. The remaining 36% showed an additional cell peak with a DNA index ranging from 1.15 to 1.92. The percentage of S-phase cells was higher in malignant (median = 3.8) than in benign tumors (median = 1.9) (p less than .001), without correlation to histological tumor subtype.

Aneuploidy↗

PubMed1985

Cytokinetic studies in acute leukemia during early induction therapy.

The proliferative activity of bone marrow blasts was determined in 19 patients with acute leukemia during early induction therapy using in vitro tritiated thymidine (3H-TdR) cytoautoradiography and propidium iodide DNA flow cytofluorometry. Following 1-3 causes of treatment, the aliquot of bone marrow blasts in 9 patients, who later achieved remission or marrow aplasia, was reduced to a greater extent that of the remaining 10 patients who failed to respond. In the first group of patients, the 3H-TdR labeling index was increased by 259-653% over the pretreatment value, whereas it was lower than 104% of the pretreatment value in all but one unresponsive patient. Leukemic blast recruitment is conceivable. It might have facilitated remission by increasing the effectiveness of the antileukemic agents administered later.

Adolescent↗