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Biomedical subjects

G Mazzini

Publications and source records attributed to G Mazzini.

At least 19 recordsLinked to original sources
PubMed2006

Effect of nitric oxide on lymphocytes from sporadic amyotrophic lateral sclerosis patients: toxic or protective role?

Markers of oxidative and nitrosative stress have been found in spinal cord, cortex, cerebrospinal fluid and plasma of patients affected by amyotrophic lateral sclerosis (ALS), a fatal disorder characterised by progressive motor neuron degeneration. In this study, we investigated the effect of the NO-releasing agent, diethylamine NONOate (NONO), on lymphocytes from patients affected by the sporadic form of ALS (SALS) and controls by flow cytometry. In the same experimental conditions we investigated the expression of the antioxidant proteins, Bcl-2 and SOD1. Incubation with NONO induced cell damage in control lymphocytes but did not further damage the already affected untreated SALS lymphocytes. The incubation with NONO induced a time-dependent decrease of Bcl-2 and SOD1 in control lymphocytes. Surprisingly, in SALS lymphocytes the NONO treatment increased the expression of these proteins, which in basal conditions was depressed compared to control lymphocytes.

Amyotrophic Lateral Sclerosis↗
PubMed2003

Dual excitation multi- fluorescence flow cytometry for detailed analyses of viability and apoptotic cell transition.

The discrimination of live/dead cells as well as the detection of apoptosis is a frequent need in many areas of experimental biology. Cell proliferation is linked to apoptosis and controlled by several genes. During the cell life, specific events can stimulate proliferation while others may trigger the apoptotic pathway. Very few methods (i.e. TUNEL) are now available for studies aimed at correlation between apoptosis and proliferation. Therefore, there is interest in developing new methodological approaches that are able to correlate apoptosis to the cell cycle phases. Recently new approaches have been proposed to detect and enumerate apoptotic cells by flow cytometry. Among these, the most established and applied are those based on the cell membrane modifications induced in the early phases of the apoptotic process. The dye pair Hoechst 33342 (HO) and Propidium Iodide (PI), thanks to their peculiar characteristics to be respectively permeable and impermeable to the intact cell membrane, seems to be very useful. Unfortunately the spectral interaction of these dyes generates a consistent "energy transfer" from HO to PI. The co-presence of the dyes in a nucleus results in a modification in the intensity of both the emitted fluorescences. In order to designate the damaged cells (red fluorescence) to the specific cell cycle phases (blue fluorescence), we have tested different staining protocols aimed to minimize the interference of these dyes as much as possible. In cell culture models, we are able to detect serum-starved apoptotic cells as well as to designate their exact location in the cell cycle phases using a very low PI concentration. Using a Partec PAS flow cytometer equipped with HBO lamp and argon ion laser, a double UV/blue excitation has been performed. This analytical approach is able to discriminate live blue cells from the damaged (blue-red) ones even at 0.05 micro g/mL PI. The same instrumental setting allows performing other multi-colour analyses including AnnexinV-FITC as well as the possibility to make a correlated analysis to phenotype markers.

Apoptosis↗
PubMed2001

Cell kinetics in a model of artificial skin. An immunohistochemical and flow cytometric analysis.

Bioengineered organs raised in vitro are candidate substitutes for natural organs in biological, pharmacological and clinical applications. We have studied cell kinetics in a human skin equivalent (HSE) using a combined immunohistochemical and flow cytometric approach. Morphological analysis has shown that, relative to unstimulated natural skin, cell proliferation mainly occurs in the basal layer of the epidermal equivalent. Immunohistochemical and flow cytometric measurements of the growth fraction suggested a cell turnover comparable to that of natural skin. Immunohistochemical labelling indices matched well with flow cytometric data. These observations are consistent with morphological and histochemical data demonstrating normal cell differentiation and tissue architecture in HSE and suggest that such HSE may be a usefull substitute for human skin.

Biocompatible Materials↗
PubMed1997

Comparison between Ki-67 index and S-phase fraction on fine-needle aspiration samples from breast carcinoma.

BACKGROUND: Fine-needle aspiration (FNA) biopsy has been used increasingly in the diagnosis and biologic characterization of breast carcinomas in patients who receive preoperative chemotherapy. Because proliferative activity of breast carcinoma has been shown to be of prognostic significance, the authors compared immunocytochemical Ki-67 growth fraction and flow cytometric S-phase fraction (SPF), both evaluated on FNA samples. METHODS: The proliferative activity of 134 FNA samples from primary breast carcinoma patients was studied using both immunocytochemistry with the monoclonal antibody Ki-67 and SPF determined by DNA flow cytometry. RESULTS: Ki-67 and SPF were evaluable in 114 and 107 cases, respectively, and both were evaluable in 95 cases. Of the 134 FNA samples studied, 37% were diploid and 63% were aneuploid. The distribution of both Ki-67 and SPF was different in diploid and aneuploid tumors. The median Ki-67 value as well as the median SPF were significantly higher in aneuploid versus diploid tumors (P < 0.001). Median Ki-67 and SPF values were used to discriminate between low versus high proliferating tumors. The overall concordance between Ki-67 and SPF was 75% (P < 0.001). A good correlation was found between Ki-67 and SPF (correlation coefficient = 0.72; P < 0.001). CONCLUSIONS: The results of the current study suggest that Ki-67 growth fraction and SPF determined by FNA may be used as measurements of the proliferative activity of breast carcinoma. The authors recommend these determinations be used as preoperative procedures in patients with a cytologic diagnosis of breast carcinoma who are candidates for neoadjuvant chemotherapy and/or endocrine therapy.

Antibodies, Monoclonal↗
PubMed1997

Quality control study of the Italian Group of Cytometry on flow cytometry DNA content measurements: II. Factors affecting inter- and intralaboratory variability.

A multicentric national quality control study has been organized under the auspices of the Italian Group of Cytometry to find a possible influence of some procedural steps in DNA flow cytometry measurements on DNA index (DI) values and to identify the main parameters affecting the interlaboratory variability. To 40 participating laboratories we provided suspensions containing unknown mixture of different cell types: an homogeneous thymocyte population used to check instrument linearity; one mixture composed of two cell types characterized by DI = 1.00 and 1.10; and another composed of three different cell types with relative DIs of 1.00, 1.26, and 1.62, respectively. Possible effects due to staining protocols were studied, allowing the participants to stain cellular DNA according to the procedure routinely adopted in each laboratory, in addition to a standardized procedure with a fixed PI solution. As far as the influence of instrument linearity on DI values is concerned, we did not find any correlation with the DI variability observed, even if the use of a standardized staining protocol could lead to a sensible gain in interlaboratory DI reproducibility. Twenty-five of 40 (65%) laboratories were able to discriminate the near-diploid subpopulation, and a coefficient of variation of less than 4% was the minimum condition necessary to recognize the DI = 1.1 population. In samples containing two aneuploid subpopulations, 25 of 35 (71.4%) laboratories showed a high reproducibility with the standard staining protocol and 22 of 38 (57.9%) with the free staining protocol. However, a sensible improvement in interlaboratory reproducibility emerged with respect to the previous trial.

Animals↗
PubMed1997

Cell proliferation in developing human dental pulp. A combined flow cytometric and immunohistochemical study.

Information concerning cell proliferation and differentiation in dental pulp may be important to understand tooth response to exogenous stimuli. Since few data concerning human dental pulp are available, we have investigated the growth fraction and the localization of proliferating cells in pulp tissue of third molars of young adult human males and females, using flow cytometry and immunohistochemistry. Flow cytometric analysis demonstrates a low proliferative activity of pulp tissue that appears to be confined to radicular pulp, as revealed by immunohistochemical detection of proliferating cells. No polyploid or aneuploid cell populations could be identified, and G2-blocked cells, if any, represented a negligible cell population. Odontoblasts, cells of the sub-odontoblastic layer, and cells of coronal pulp were found to be not proliferating under normal conditions. These data provide the basis for future investigations on proliferative activity and regenerative potentiality of human pulp cells in experimental and clinical situations.

Adolescent↗
PubMed1997

Automation of human sperm cell analysis by flow cytometry.

Semen sample analysis is routinely performed by microscopical evaluation and manual techniques by laboratory operators; the analysis is affected by a wide imprecision related to variability among observers, influencing its clinical validity. Our aim was to automate sperm analysis with the use of flow cytometry for evaluation of cell counts and typing and with the use of a new membrane-permeant nucleic acid stain for evaluation of sperm viability. Statistical analysis of the comparison between manual and automated methods for sperm counts was performed by the Bland and Altman method; the mean difference was 0.243 x 10(6) sperms/ mL. The precision of the flow cytometric analysis was evaluated with whole sperm; the between-run CV was 7.5% and the within-run CV was 2.5%. Data observed suggest that flow cytometric sperm analysis, with high precision and accuracy and low costs, can be proposed for routine use in clinical laboratories.

Adolescent↗
PubMed1996

Nuclear grading and flow cytometric DNA pattern in fine-needle aspirates of primary breast cancer.

Fine-needle aspiration (FNA) biopsy is increasingly used in the diagnosis and biological characterization of breast carcinomas in patients who receive preoperative chemotherapy. In this context, nuclear cytologic grade supplemented by DNA content could play an important role in the morphologic assessment of breast cancer. In this study, DNA ploidy pattern, analyzed by flow cytometry on FNAs from 92 primary breast carcinomas, was related to cytologic nuclear grade. Twenty-seven samples were cytologic grade 1, 33 were grade 2, and 32 were grade 3. Ploidy correlated with cytologic nuclear grade (P = 0.0001). Thirty percent of grade 1, 55% of grade 2, and 84% of grade 3 tumors were DNA aneuploid. For 30 of the 92 FNAs, it was possible to compare nuclear cytologic grade with the corresponding histologic grade using the Scarff, Bloom, and Richardson system. A high concordance (80%) between nuclear grade on FNAs and histologic grade was found. DNA flow cytometry in combination with nuclear cytologic grade might represent additional information for the characterization of breast cancer diagnosed by FNA.

Biopsy, Needle↗
PubMed1996

Cell proliferation and ploidy of human solid tumours: methodological experience with in vivo bromodeoxyuridine and DNA flow cytometry.

A sequential procedure for single and multiparameter flow cytometry (FCM) that allows detailed cell proliferation and DNA ploidy studies of human solid tumours is reported here. This description includes time collection and storage, tissue disaggregation and staining as well as FCM analysis of samples. The overall feasibility, together with some critical aspects of the DNA/bromodeoxyuridine (BrdU) in vivo assay for cell kinetic studies in human solid tumours, are reported, based on our experience in recent years. We found that the BrdU in vivo method, coupled with bivariate FCM for measurements, is a valuable approach for a 'dynamic' assessment of tumour cell proliferation in the clinical setting. Routine measurements can be achieved providing that well standardised tissue disaggregation and immunolabelling procedures are performed. In different solid tumours, cytokinetic results were satisfactory in 85% of cases as far as the BrdU-labelling index is concerned while 70% were acceptable for DNA synthesis time and tumour potential doubling time. Causes of failure are also considered and discussed. In order to guarantee the finest detection of aneuploidy, routine high resolution single-parameter DNA analysis is suggested. Our experience with further improvements in cell kinetic studies based on the possibility of gating the BrdU/DNA analysis for a subpopulation of interest (i.e. cytokeratin-positive cells in epithelial tumours) is also reported.

Bromodeoxyuridine↗
PubMed1995

Cell kinetics of CD34-positive hematopoietic cells following chemotherapy plus colony-stimulating factors in advanced breast cancer.

Bone-marrow (BM) hematopoietic precursors are recruited into proliferative activity when colony-stimulating factors (CSF) are sequenced with chemotherapy (CT). Previous studies suggested that further CT can be safely administered only when the increased proliferative activity of these cells has subsided, because most cytostatic drugs selectively damage cycling cells. The safest interval between CSF discontinuation and the start of the next CT course needs to be ascertained in vivo. Thirty patients with advanced breast cancer were treated with an intensified FEC regimen, planned at 21-day intervals, sequenced with granulocyte-macrophage (GM)-CSF (15 patients) or granulocyte (G)-CSF (15 patients). Using flow cytometry (FCM) we evaluated the proliferation kinetics of CD34+ BM hematopoietic progenitors before CT+CSF and at different times after CSF administration was stopped. FEC+GM- and FEC+G-CSF sequences both induced a rapid and sustained increase in the percentage of BM myeloid precursors (BMMP%) and in the cycling status of CD34+BM cells. However, while the BMMP% remained elevated in both cases after CSF were stopped, the enhanced proliferative activity of CD34+ cells decreased more rapidly after GM- than after G-CSF. Using FCM, CD34+ BM-derived hematopoietic presursor cell kinetics is readily evaluated in the clinical setting. The administration of CSF following CT increases both the proliferative activity of CD34+ BM cells and the BMMP%. After CSF were discontinued a kinetic refractoriness of hematopoietic progenitors was more evident after GM-CSF than after G-CSF. These data may be of value in designing clinical trials to avoid cytostatic damage to the BM hematopoietic stem-cell compartment.

Aged↗
PubMed1995

Oligodeoxynucleotides antisense to c-abl specifically inhibit entry into S-phase of CD34+ hematopoietic cells and their differentiation to granulocyte-macrophage progenitors.

A number of experimental observations suggest that the proto-oncogene c-abl participates in the regulation of hematopoietic cell growth. We used an antisense strategy to study the relationship between c-abl expression and hematopoietic cell proliferation and differentiation. Purified normal human bone marrow-derived CD34+ cells were obtained by immunomagnetic selection and incubated with 18-base-unmodified antisense oligodeoxynucleotides complementary to the first six codons of the two alternative first exons of c-abl, la and lb. At the end of incubation, an aliquot of cells was assayed for clonogenic growth and the remainder was used for flow cytometric analyses. Cell kinetics were evaluated by means of both single parameter DNA and bivariate DNA/bromodeoxyuridine (BrdU) flow cytometry. Apoptosis was routinely studied by DNA flow cytometric analysis and, in some cases, also through DNA agarose gel electrophoresis for detection of oligonucleosomal DNA fragments. Expression of differentiation markers was studied by flow cytometry. Exposure to antisense oligonucleotides specifically inhibited the accumulation of c-abl mRNA in CD34+ cells. Preincubation with the c-abl antisense oligomers reduced the proportion of cells in S-phase from 19% +/- 5% (mean +/- SD) to 7% +/- 4% (P < .05), and BrdU labeling from 13% +/- 6% to 6% +/- 3% (P < .05). Flow cytometry and DNA agarose gel electrophoresis showed that treated CD34+ cells accumulated in the G0/G1 region of the DNA histogram with no evidence of either differentiation or apoptosis. By contrast, both growth factor deprivation and exposure of CD34+ cells to the tyrosine kinase inhibitor tyrphostin AG82 clearly induced apoptosis. When cells were preincubated with antisense oligonucleotides and then plated for evaluation of colony formation, this resulted in a significant inhibition of colony forming unit granulocyte-macrophage growth (from 44 +/- 15 to 22 +/- 9; P < .01) but had no effect on burst-forming unit erythroid growth (24 +/- 11 v 21 +/- 11; P < .05). These results suggest that c-abl expression is critical for entry of human CD34+ hematopoietic cells into S-phase and for their differentiation to granulocyte-macrophage progenitors. They also indicate that other tyrosine kinases besides p145c-alb are active in the prevention of apoptosis, so that inhibition of c-abl RNA accumulation arrests CD34+ cells in G0/G1 without activating programmed death.

Antigens, CD34↗
PubMed1995

In vivo enhanced antitumor activity of carmustine [N,N'-bis(2-chloroethyl)-N-nitrosourea] by simvastatin.

The effects of a combination of simvastatin, a cholesterol-lowering agent, and carmustine (BCNU; N,N'-bis(2-chloroethyl)-N-nitrosourea) on experimental C6 glioma were studied in vitro and in vivo. In vitro simvastatin and BCNU alone inhibited cell proliferation in a dose-dependent fashion. A subliminal concentration of simvastatin (0.1 microM) markedly and synergistically increased the BCNU toxicity to C6 glioma cells. The cytofluorimetric analysis of DNA from simvastatin-treated C6 glioma cells showed, besides the already described arrest in G1, an arrest/retardation in G2-M. Mitotic index from C6 cells incubated with simvastatin (10 microM) decreased by about 90%, indicating a specific C6 arrest/retardation in G2. The drug effects could be completely reversed by simvastatin withdrawal or mevalonate addition to the cultured cells. The combination of simvastatin and BCNU resulted predominantly from the profound retardation of cells in the G2-M compartment of the cell cycle. In vivo simvastatin (administered daily mixed with food) and BCNU (single i.p. injection), when given separately, caused a dose-dependent inhibition of labeling index in C6 glioma homografts (ID50, 61 mg/kg/day and 8.7 mg/kg, respectively). The combination of the lowest doses tested (simvastatin, 25 mg/kg/day and BCNU 0.3 mg/kg) resulted in a significant growth delay (compared to either drug alone) in C6 glioma (P < 0.05). There was no significant increase in toxicity as assessed by myelosuppression (WBC counts and bone marrow labeling index) and body weight. The results provide in vivo support for the combined use of simvastatin, a cholesterol-lowering agent, and BCNU in brain tumor treatment.

Animals↗
PubMed1995

Detection of bromo-deoxyuridine- and proliferating cell nuclear antigen-immunoreactivities in tooth germ.

The development of antibodies to cell cycle-related antigens provides the basis for immunochemical studies on cell kinetics. Bromo-deoxyuridine (BrdU) incorporated by S-phase traversing cells is an exogenous marker of replicating cells, whereas proliferating cell nuclear antigen (PCNA) is an endogenous marker of replicating cells. We have applied monoclonal antibodies to BrdU and PCNA to study cell kinetics in tooth germ by immunohistochemistry and flow cytometry. BrdU-antibody reacted only with S phase-traversing cells in pulse-labelling experiments, whereas PCNA-antibody reacted with G1, S and G2-M phases traversing cells. Although the number of PCNA-positive cells largely exceeded the number of BrdU-labelled cells, the pattern distribution of immunoreactive cells was similar using BrdU- and PCNA-antibodies as revealed by immunohistochemistry. The use of PCNA-antibody allowed the detection of proliferating cells also in human tooth germ. It is suggested that combined identification of BrdU and PCNA on one side and growth factors, oncoproteins or differentiation markers on the other side may constitute a useful approach to understand the mechanisms of cell differentiation in tooth germ.

Animals↗
PubMed1995

High dose intravenous immune globulins and plasma exchange in Guillain-Barré Syndrome.

We report the effects of treatment with plasma-exchange (PE) and intravenous immune globulins (IVIg) in 36 out of 50 patients with Guillain-Barré syndrome (GBS) recruited by an incidence study in the Emilia-Romagna region of Italy. Comparison of the patients treated with PE and IVIg showed no significant differences in terms of effectiveness in improving the clinical course of GBS: at one month, respectively 11.1% and 25% had recovered, and 55.5% and 58.3% had improved by at least one grade. These results are in agreement with those of the Dutch GBS trial. No relapses were observed in either group. Moreover, our results showed no difference in clinical outcome at 1 and 3 months between the patients receiving only one therapy and those receiving two; a second cycle of therapy did not seem to improve the clinical course of the disease significantly. We conclude that PE and IVIg are both safe and effective therapies for GBS.

Adolescent↗
PubMed1994

Cell kinetics of human ovarian cancer with in vivo administration of bromodeoxyuridine.

BACKGROUND: Cell kinetics could have prognostic significance in human ovarian cancer and might also help in designing optimal therapy. No data are available on the in vivo kinetics of this tumor using bromodeoxyuridine (BrdU) infusion before surgery. PATIENTS AND METHODS: The kinetic parameters of human ovarian carcinoma were investigated in vivo using BrdU incorporation and bivariate BrdU/DNA flow cytometric (FCM) analysis. Fifty-five previously untreated patients with ovarian cancer (F.I.G.O. clinical stage III and IV) were studied. BrdU (250 mg/sqm) was given i.v. 6 h before surgery and samples of primary tumor and metastasis biopsies were fixed in 70% ethanol. By coupling the BrdU immunoreaction with biparametric FCM analysis, the nuclear DNA content (i.e., ploidy status), the tumor labelling index (LI), the synthesis time (TS) and the potential doubling time of the tumor mass (Tpot) were obtained. BrdU immunodetection was done on histological sections of the same tumors. RESULTS: The majority of the tumors had a DNA aneuploid content (71.5%). The amount of BrdU-positive S-phase cells varied in different tumor samples and when several samples were taken from the same tumor. The proportion of BrdU-negative S-phase cells was large (50% of the total S phase cells) in almost all cases. The mean LI was 6.1% using FCM and 7.2% on visual count of the slide. The mean TS and Tpot were 14.7 h and 12.5 days, respectively. LI and TS were not correlated with clinical tumor stage, histological grading, residual tumor size or DNA ploidy, but Tpot was significantly higher (p < 0.05) in patients with residual tumor size < 2 cm. Univariate analysis showed that Tpot was significantly associated with the response after first-line chemotherapy (p < 0.009). In multivariate analysis only residual tumor size was related to the response. CONCLUSION: Although this in vivo BrdU technique provides information on the kinetic features of human ovarian cancers, it remains questionable whether this information has any additional value compared to currently used prognostic factors which are assessable clinically and surgically.

Analysis of Variance↗
PubMed1993

Cell cycle kinetic effects of tamoxifen on human breast cancer cells. Flow cytometric analyses of DNA content, BrdU labeling, Ki-67, PCNA, and statin expression.

Tamoxifen is known to inhibit the growth of some human mammary carcinoma cells; this effect is accompanied by a decrease in the proportion of cells synthesizing DNA. In this work, flow cytometry of DNA and of bromodeoxyuridine labeling and the evaluation of the cell cycle-related antigens Ki-67, PCNA, and statin were used to investigate the changes in the proliferation kinetics of MCF-7 cells before and after treatment with 10(-7) M TAM. The treatment with TAM induced a significant decrease in the fraction of S-phase cells and an increase in those with a DNA content typical of G0/1 phase. The TAM-induced block in G0/1 is paralleled by a decrease in the frequency of cells expressing Ki-67 and PCNA, and by an increase in statin-positive (G0) cells. These results confirmed that the TAM-induced inhibition of cell growth is associated with major changes in the cell cycle parameters of MCF-7 cells, and provide the first experimental evidence that two main mechanisms are operating: the accumulation of cells in G1, before the onset of S-phase, and the exit of some cells from the cycling compartment.

Biomarkers, Tumor↗