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Biomedical subjects

R Invernizzi

Publications and source records attributed to R Invernizzi.

At least 19 recordsLinked to original sources
PubMed2006

Flow cytometry evaluation of erythroid dysplasia in patients with myelodysplastic syndrome.

Erythroid dysplasia is the pathologic hallmark of myelodysplastic syndromes (MDS). To develop a quantitative flow-cytometry approach to its evaluation, we analyzed the expression of CD71, CD105, cytosolic H-ferritin (HF), cytosolic L-ferritin (LF) and mitochondrial ferritin (MtF) in erythroblasts from 104 MDS patients, 69 pathologic control patients and 19 healthy subjects. Six-parameter, 4-color flow cytometry was employed, and data were expressed as mean fluorescence intensity. Compared with pathologic and healthy controls, MDS patients had higher expression of HF (P < 0.001) and CD105 (P < 0.001), and lower expression of CD71 (P < 0.001). MtF was specifically detected in MDS with ringed sideroblasts, and there was a close relationship between its expression and Prussian blue staining (r = 0.89, P < 0.001). In vitro cultures of myelodysplastic hematopoietic progenitors showed that both HF and MtF were expressed at a very early stage of erythroid differentiation, and that MtF expression is specifically related to mitochondrial iron loading. A classification function based on expression levels of HF, CD71 and CD105 allowed us to correctly classify > 95% of MDS patients. This flow-cytometry approach provides an accurate quantitative evaluation of erythroid dysplasia and allows a reliable diagnosis of sideroblastic anemia, and may therefore be a useful tool in the work-up of patients with MDS.

Adult↗
PubMed2005

Flow cytometry evaluation of erythroid and myeloid dysplasia in patients with myelodysplastic syndrome.

The purpose of this study was to develop a flow cytometric approach to the evaluation of marrow dysplasia in myelodysplastic syndromes (MDS). We first studied a cohort of 103 MDS patients as well as 46 pathological and healthy controls. Flow cytometry data were expressed as percentage of positive cells. Analysis of erythroid cells showed higher proportions of immature cells (P < 0.001) and decreased levels of CD71 expression on nucleated red cells (P = 0.02) in MDS. Analysis of myeloid cells showed lower proportions of CD10+ and higher proportions of CD56+ granulocytes (P < 0.001), and increased ratios of immature to mature cells (P = 0.007). Since no single immunophenotype could accurately differentiate MDS from other conditions, we used discriminant analysis for generating erythroid and myeloid classification functions using combinations of immunophenotypic parameters. These functions were prospectively validated in a testing cohort of 69 MDS patients and 46 pathological controls. A diagnosis of MDS was obtained in 60/69 cases (87%). No false-positive results were noticed among controls. Significant correlations between values of these functions and both degree of morphological dysplasia and the International Prognostic Scoring System were found. These findings indicate that flow cytometry evaluation of marrow dysplasia is feasible and may be useful in the work-up of individual MDS patients.

Antigens, CD34↗
PubMed2004

Impaired bone marrow hematopoietic progenitor cell function in rheumatoid arthritis patients candidated to autologous hematopoietic stem cell transplantation.

We have evaluated bone marrow morphology, percentage of bone marrow CD34(+) cells, proliferative activity of bone marrow precursors, clonogenic assay (BFU-E and CFU-GM) in short-term bone marrow cultures, and bone marrow cell apoptosis, together with serum TNF-alpha and IL-6, in 16 chronic, refractory RA patients, as well as in five healthy controls. Of 16 RA patients (68.7%), 11 showed a reduced bone marrow cellularity, while it was normal in all the controls. In RA patients, the median percentage of CD34(+) bone marrow cells, the median percentage of proliferating bone marrow myeloid precursors, and the median number of both BFU-E and CFU-GM colonies were significantly lower than observed in the controls. As far as TNF-alpha and IL-6 titers is concerned, the latter did not significantly differ from controls' values, while TNF-alpha titers were significantly lower in healthy controls. Finally, the median apoptotic index of early bone marrow myeloid cells of RA patients was significantly higher compared with controls. These observations may identify the biological risk factors for impaired mobilization and/or engraftment when RA patients are candidates for autologous hematopoietic stem cell grafting.

Adult↗
PubMed2004

Retrospective comparison of qualitative and quantitative reverse transcriptase polymerase chain reaction in diagnosing and monitoring the ALL1-AF4 fusion transcript in patients with acute lymphoblastic leukaemia.

We compared quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) to qualitative RT-PCR in determining response to therapy and predicting clinical outcome in 18 retrospectively selected patients with ALL positive for the ALL1-AF4 fusion and with frozen RNA samples collected at diagnosis and during follow-up (96 samples analysed). The ALL1-AF4 junction was detected by qualitative RT-PCR in 18 patients and by Q-RT-PCR in 17 patients (one patient harboured the rare e10-e6 ALL1-AF4 junction, which falls outside of the primer and probe location designed for the Q-RT-PCR). In three of the 12 patients negative to qualitative RT-PCR after induction therapy, a small number of ALL1-AF4 copies was detected by Q-RT-PCR. Thus nine patients were negative and eight positive. Seven of the eight positive patients suffered a relapse, including two of the three patients positive to Q-RT-PCR yet negative to qualitative RT-PCR. Moreover, we found two (5%) discordant results among the 39 follow-up tests of the nine patients who converted to a negative qualitative-quantitative PCR status. The results suggest that qualitative RT-PCR is more appropriate for the routine diagnosis of this genetic alteration. However, Q-RT-PCR is more accurate in assessing the molecular response after induction treatment and could be more useful in clinical decision-making in ALL1-AF4-positive ALL patients.

Adolescent↗
PubMed2001

A human mitochondrial ferritin encoded by an intronless gene.

Ferritin is a ubiquitous protein that plays a critical role in regulating intracellular iron homoeostasis by storing iron inside its multimeric shell. It also plays an important role in detoxifying potentially harmful free ferrous iron to the less soluble ferric iron by virtue of the ferroxidase activity of the H subunit. Although excess iron is stored primarily in cytoplasm, most of the metabolically active iron in cells is processed in mitochondria. Little is yet known of how these organelles regulate iron homeostasis and toxicity. Here we report an unusual intronless gene on chromosome 5q23.1 that encodes a 242-amino acid precursor of a ferritin H-like protein. This 30-kDa protein is targeted to mitochondria and processed to a 22-kDa subunit that assembles into typical ferritin shells and has ferroxidase activity. Immunohistochemical analysis showed that it accumulates in high amounts in iron-loaded mitochondria of erythroblasts of subjects with impaired heme synthesis. This new ferritin may play an important role in the regulation of mitochondrial iron homeostasis and heme synthesis.

Amino Acid Sequence↗
PubMed2001

Clinical and biological features of acute myeloid leukaemia occurring as second malignancy: GIMEMA archive of adult acute leukaemia.

Between July 1992 and June 1996, 3934 new cases of acute leukaemia were registered in the Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto (GIMEMA) Archive of Adult Acute Leukaemia. Two hundred cases (5.1%) presented with a history of primary malignancy (PM), 179 of which were acute myeloid leukaemia (AML). The median age of these cases was significantly higher than that of other primitive AML (63 years vs. 57 years; P < 0.001). The number of men was significantly lower than the number of women [74/1544 (4.8%) vs. 105/1420 (7.4%); odds ratio (OR) 0.63, 95% confidence interval (CI) 0.46-0.87; P < 0.002], as was the number of patients aged <65 years [104/1963 (5.3%) vs. 75/1001 (7.5%); OR 0.69, 95% CI 0.50-0.95; P < 0.01]. An increased incidence of cancer was observed among first-degree relatives of patients with AML occurring after a PM (secondary AML; sAML) [66/179 (36.9%) sAML vs. 757/2785 (27.2%) de novo AML, age adjusted; OR 2.62, 95% CI 1.07-6.42; P < 0.005]. Prevalent types of PM were breast cancer, lymphoma and Hodgkin's disease. sAML occurred after a median latency of 52 months (range 2-379). Of the 122 patients who received chemotherapy for sAML, 67 patients (55%) achieved a complete remission (CR), three a partial remission, 15 (12%) died in induction and 37 (30%) were unresponsive. The median duration of CR was 30 weeks (range 4-250). The median overall survival was 7 months (range 1-196). Comparing acute promyelocytic leukaemia with all other French-American-British (FAB) groups, a significant increase in CR achievement was observed [14/18 (77.7%) vs. 53/101 (52.4%), P < 0.046] as well as in median CR duration (55 vs. 24 months, P < 0.02). The analysis of our data suggests that not only previous chemotherapy but also genetic predisposition could play a role in the pathogenesis of sAML.

Acute Disease↗
PubMed2001

A modified high-dose dexamethasone regimen for primary systemic (AL) amyloidosis.

High-dose dexamethasone (HD-Dex) has been reported to benefit AL amyloidosis patients with varying response rates. Our preliminary experience with the usual HD-Dex schedule indicated that the induction phase was rather toxic in AL patients. We therefore adopted a milder schedule consisting of dexamethasone 40 mg on d 1-4 q21 d for up to eight cycles. Overall 8 out of 23 (35%) treated patients responded to treatment in a median time of 4 months (range 2-6 months) without significant toxicity. This regimen may be considered front-line therapy when autologous stem cell transplantation is not feasible and when a rapid response is particularly important.

Adult↗
PubMed2001

Haemolytic onset of Wilson disease in a patient with homozygous truncation of ATP7B at Arg1319.

We describe a 19-year-old woman with haemolytic anaemia and thrombocytopenia as the initial manifestation of Wilson disease (WD). There are two reasons for reporting such an improbable case. First, it emphasizes the importance of recognizing atypical clinical presentations of potentially lethal recessive traits for which therapy is available. Second, it shows that, even in a monogenic disorder like WD, the phenotype cannot be extrapolated from the mutated genotype in a simple fashion; this patient had a relatively late-onset form of WD despite homozygosity for a genetic lesion leading to an apparent complete loss of function of the WD copper transporter.

Adenosine Triphosphatases↗
PubMed2001

Expression of p53, bcl-2 and ras oncoproteins and apoptosis levels in acute leukaemias and myelodysplastic syndromes.

We analysed by immunocytochemistry the expression of p53, bcl-2 and ras proteins in bone marrow blasts from 59 patients with acute leukaemia (AL), 36 myeloid (AML) and 23 lymphoid (ALL), and from 22 patients with myelodysplastic syndrome (MDS); our aim was to examine if abnormalities in their expression were associated with peculiar biological and clinical findings, or with an altered apoptosis rate, as measured by TUNEL technique. The oncoproteins were expressed with extreme variability, without significant differences among the various morphological or immunological AL subtypes. The mean percentages of bcl-2+ blasts were significantly higher in AML than in MDS (p = 0.01), and in MDS with bone marrow blastosis than in the forms without excess of blasts (p = 0.007). The lowest percentages of apoptotic cells were observed in ALL (mean 1%, p = 0.006), whereas in MDS the apoptotic index was higher (16.7%) than in AML (8.6%) and than in the normal controls (10.8%). but the difference tended to be statistically significant only for cases of refractory anaemia. Whereas in AML and MDS the apoptotic rate was independent of the oncoprotein expression, in ALL there was a significant linear relationship between TUNEL and ras positivity (p = 0.01). Among AML patients treated with intensive polychemotherapy, no differences were observed in oncoprotein expression and apoptotic rate between responders and resistant cases. In conclusion, our data are in agreement with the hypothesis that decreased apoptosis and enhanced cell survival are associated with AL, whereas a high level of apoptosis may be responsible for the ineffective hematopoiesis in MDS; abnormal expression of oncoproteins, even if not strictly related to apoptosis level, may influence disease behaviour.

Acute Disease↗
PubMed2000

JL13, a pyridobenzoxazepine compound with potential atypical antipsychotic activity, increases extracellular dopamine in the prefrontal cortex, but not in the striatum and the nucleus accumbens of rats.

In behavioral and receptor binding studies, 5-(4-methylpiperazin-1-yl)-8-chloro-pyridol[2,3b] [1,5]benzoxazepine (JL13) shows an atypical antipsychotic profile. We used microdialysis in awake rats to study the effects of various intraperitoneal doses of JL13 on extracellular concentrations of dopamine in the prefrontal cortex, nucleus accumbens and striatum. JL13 at 20 mg/kg and 40 mg/kg dose-dependently raised extracellular dopamine (234% and 434% of basal levels at peak, respectively) in the prefrontal cortex whereas lower doses (5 mg/kg and 10 mg/kg) had no effect. Extracellular concentrations of dihydroxyphenylacetic acid and homovanillic acid were also significantly increased in the prefrontal cortex of rats given 40 mg/kg JL13 (310% and 230% of basal levels, respectively). At 20 mg/kg and 40 mg/kg JL13 did not affect the extracellular concentrations of dopamine and its metabolites in the striatum and nucleus accumbens. The mechanisms by which JL13 increases cortical dopamine release and the significance for potential antipsychotic efficacy are discussed.

3,4-Dihydroxyphenylacetic Acid↗
PubMed2000

Cell cycle distribution of cord blood-derived haematopoietic progenitor cells and their recruitment into the S-phase of the cell cycle.

The objective of this study was to evaluate the cycling status of cord blood (CB)-derived colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC), and their recruitment into the S-phase of the cell cycle. By using the cytosine arabinoside (Ara-C) suicide approach, we found that only small proportions of both CFC and LTC-IC were in the S-phase of the cell cycle. These estimates were confirmed by flow cytometric DNA analysis, which showed that 96 +/- 2% of CB-derived CD34+ cells were in G0/G1 and only 1.6 +/- 0.4% in the S-phase. Staining of CD34+ cells with an antistatin monoclonal antibody, a marker of the G0 phase, indicated that among CD34+ cells with a flow cytometric DNA content typical of the G0/G1 phase 68 +/- 7% of cells were in the G0 phase of the cell cycle. Incubation (24 h) with interleukin 3 (IL-3), recombinant human stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) significantly increased the proportion of cells in the S-phase for both CFC and LTC-IC without inducing any loss in numbers. Flow cytometric DNA analysis also showed an increase in CD34+ cells in the S-phase upon continuous exposure to these cytokines. Our findings indicate that: (i) very few CB-derived CFC or LTC-IC were in the S-phase of the cell cycle; (ii) a substantial amount of CD34+ cells with a flow cytometric DNA content typical of the G0/G1 fraction was cycling, as found in the G1 phase of the cell cycle; and (iii) 24-h incubation with IL-3, SCF and G-CSF could drive a proportion of progenitor cells into the S-phase without reducing their number. These data might be useful for gene transfer protocols and the ex vivo expansion of CB-derived progenitor cells.

Antigens, CD34↗
PubMed2000

Alpha-synuclein and Parkinson's disease: selective neurodegenerative effect of alpha-synuclein fragment on dopaminergic neurons in vitro and in vivo.

Missense mutations in the alpha-synuclein gene were associated with a familial Parkinson's disease, and alpha-synuclein is a major component of Lewy bodies, the intracellular inclusions that neuropathologically characterize Parkinson's disease. We investigated the neurotoxic activity of the nonamyloid component (NAC) of senile plaque, the fibrillogenic fragment (61-95) of alpha-synuclein, in vitro and in vivo. Rat primary mesencephalic neurons were exposed for 6 days to low concentrations of preaggregated NAC (0.5-10.0 microM). The number of dopaminergic neurons and dopamine content were both reduced with no effect on the general viability of the cells. At higher concentrations (25-100 microM), the neurotoxic effect of NAC was extended to all neurons. Preaggregated NAC was also toxic on a PC12 dopaminergic cell line differentiated with nerve growth factor. The intracellular localization of NAC has been identified by the exposure of neuronal cells to fluorescent peptide. In vivo application of aggregated NAC in the substantia nigra induced loss of dopaminergic neurons. Our data illustrate the selective neurotoxic effect of NAC for dopaminergic neurons and support the central role of alpha-synuclein in the pathogenesis of Parkinson's disease.

Animals↗
PubMed2000

Cord blood-derived hematopoietic progenitor cells: in vitro response to hematopoietic growth factors and their recruitment into the S-phase of the cell cycle.

BACKGROUND AND OBJECTIVES: In the recent years many studies on the expansion of cord blood (CB)-derived progenitor cells have been performed, whereas less information is available on their cycling status. The objective of this study was to evaluate the cycling status of CB-derived colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC), and their recruitment into the S-phase of the cell cycle in response to a combination of cytokines. DESIGN AND METHODS: CB-derived CFC and LTC-IC were first quantified by standard clonogenic assay and long-term culture, respectively. In a second set of experiments, CB-derived progenitor cells were incubated with interleukin(IL)-3, stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) and their cell cycle status assessed both by the cytosine arabinoside (Ara-C) suicide approach and by flow cytometric DNA analysis. RESULTS: We found that only small proportions of both CFC and LTC-IC were in the S-phase of the cell cycle. These estimates were confirmed by flow cytometric DNA analysis, which showed that 96% +/- 2% of CB-derived CD34+ cells were in G0/G1 and only 1.6% +/- 0.4% in the S-phase. Staining of CD34+ cells with an anti-statin monoclonal antibody, a marker of the G0 phase, indicated that among CD34+ cells with a flow cytometric DNA content typical of the G0/G1 phase, 68% +/- 7% of cells were in the G0 phase of the cell cycle. Twenty-four hour incubation with IL-3, SCF and G-CSF significantly increased the proportion of cells in the S-phase for both CFC and LTC-IC without inducing any loss in their number. Flow cytometric DNA analysis also showed an increase of CD34+ cells in the S-phase upon continuous exposure to these cytokines. INTERPRETATIONS AND CONCLUSIONS: Our findings indicate that: i) a small number of CB-derived CFC and LTC-IC are in the S-phase of the cell cycle; ii) a substantial number of CD34+ cells with a flow cytometric DNA content typical of the G0/G1 fraction are cycling, as they are found in the G1 phase of the cell cycle; iii) 24-hour incubation with IL-3, SCF and G-CSF can drive a proportion of progenitor cells into the S-phase without reducing their number.

Cells, Cultured↗
PubMed1999

Acute lymphoblastic leukaemia occurring as second malignancy: report of the GIMEMA archive of adult acute leukaemia. Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto.

Between July 1992 and June 1996, 901 new cases of adult acute lymphoblastic leukaemia were recorded in the GIMEMA Archive of Adult Acute Leukaemia; 21 of them (2.3%) had a previous primary malignancy (PM). We found that secondary acute lymphoblastic leukaemia cases (sALL) presented with older age, a high incidence of pre-pre-B immunophenotype and a significantly higher prevalence of cancer among relatives compared to de novo ALL. The leukaemogenic activity of the cytotoxic drugs employed for the treatment of PM may have played a potential role in only a proportion of patients, opening the possibility that some sALL patients may have developed two or more malignancies due to individual predisposing factors.

Adolescent↗