Bivariate flow cytometric analysis of DNA content versus immunopositivity for ribonucleotide reductase M1 subunit in the cell cycle.

Ribonucleotide reductase (RR) is a cytoplasmatic enzyme catalyzing the reduction of all four ribonucleotides to their corresponding deoxyribonucleotides. Its activity strongly correlates to the rate of DNA synthesis. By using a specific monoclonal antibody against the large M1 subunit of RR, we assessed the expression of M1-RR versus DNA content by dual-parameter flow cytometry. The aim of this paper was to compare the variations in the immunopositivity for M1-RR during the cell cycle to the positivity for other cell cycle markers identifying either proliferating cells (Ki-67 and PCNA) or quiescent cells (statin). To do this, normal human embryonic fibroblasts in different growth conditions as well as several other mammalian cell lines (rat C6 glioma cells; mouse 3T3 fibroblasts and B16 melanoma cells; human epithelial EUE cells and mammary carcinoma MCF-7 cells) were used. The expression of M1-RR antigen was found to correlate positively with the expression of Ki-67 and PCNA, and negatively with the expression of statin. During early G1 phase, M1-RR becomes detectable by specific antibodies relatively later compared to PCNA and Ki-67; therefore, the lack of immunopositivity for M1-RR cannot be taken as an absolute indication of cell quiescence in G0.

All-trans retinoic acid (ATRA)-induced apoptosis is preceded by G1 arrest in human MCF-7 breast cancer cells.

In this study the effects of all-trans retinoic acid (ATRA) on cell cycle and apoptosis of MCF-7 human breast cancer cells were investigated to elucidate the mechanisms underlying the antineoplastic potential of this retinoid in breast cancer. The antiproliferative effect of ATRA was evaluated by DNA content measurements and dual-parameter flow cytometry of bromodeoxyuridine (BrdU) incorporation and of the expression of cell cycle-related proteins (Ki-67 as proliferation marker and statin as quiescence marker) vs DNA content. Apoptosis was also studied by flow cytometry of either DNA content or Annexin V labelling. After 10(-6) M ATRA treatment, the fraction of S-phase cells decreased significantly, and cells accumulated in the G0/G1 range of DNA contents. Dual-parameter flow cytograms showed a decrease in the percentage of Ki-67-labelled cells (after 10 days, only 20% of the cells were still positive for Ki-67 compared with 95% in controls), while the fraction of statin-positive cells increased slightly. From 3 days of treatment onwards, apoptosis was found to occur. These results show that ATRA-induced inhibition of MCF-7 cell growth is related to two mechanisms, i.e. the block of cell proliferation, mostly in a pre-S phase, and the induction of apoptosis. These results should be taken into account when attempting to design treatment programmes that associate ATRA with antineoplastic compounds of different cell cycle specificity.

The expression of proliferation and quiescence associated antigens in acute myeloid leukemia correlates with survival duration: analysis of 15 refractory cases.

In this study, blast cells from 15 patients with acute myeloid leukemia resistant to induction therapy were examined with two monoclonal antibodies that identify, respectively, the nuclear protein specifically expressed in non proliferating cells (statin) and the proliferating cell nuclear antigen (PCNA). We found that statin values varied widely, ranging from 0.6% to 14.7% (mean value 6.4%). When the patients were subdivided according to the mean value, those presenting with higher statin values survived for a shorter period of time than the ones characterized by lower levels (p = 0.003). We observed a wide variation in the range of PCNA values; however, if an agreement between survival duration and at least one of the proposed markers was considered, all but one case displayed concordance between survival duration and PCNA and/or statin values (in addition, 4/15 cases showed agreement for both markers). These preliminary data could indicate a possible discriminating prognostic factor between categories of patients characterized by different aspects of resistance, perhaps susceptible to different salvage therapy approaches.

Identification of resting cells by dual-parameter flow cytometry of statin expression and DNA content.

Statin, a 57-kDa nuclear protein, has been recognized as a unique marker of quiescent (G0) cells; specific monoclonal antibodies (MoAb) against statin have been produced and used to label resting cells in tissue sections and in cultured cells. We present an improved method for the identification of G0 cells by dual-parameter flow cytometry of statin expression and DNA content. The appropriate technical conditions were set up by using resting and cycling human fibroblasts as a model cell system. Several fixatives proved to be suitable for the immunocytochemical detection of statin; among them, 70% ethanol was selected because this fixation procedure is suitable for DNA staining with intercalating dyes and is routinely used for the immunolabeling of proliferation markers (such as proliferating cell nuclear antigen [PCNA] and Ki-67) and of bromodeoxyuridine (BrdUrd) incorporation. Following cell permeabilization with detergent, exposure to the antistatin antibody (S-44), and indirect fluorescein isothiocyanate immunolabeling, cells were counterstained for DNA with propidium iodide and analyzed by dual-parameter flow cytometry. In cells from several animal sources (rat thymocytes and C6 glioma cells, mouse 3T3 cells, and human MCF-7 cells), under different experimental conditions, the expression of statin was found to correlate inversely with that of PCNA and Ki-67, and with the BrdUrd labeling index. In dual-parameter flow scattergrams, G0 (statin positive) cells can be discriminated from the potentially cycling (statin negative) G1 cells, i.e., within a cell fraction having the same DNA content. This approach can be envisaged as a powerful tool both for monitoring changes in the resting cell fraction and for investigating the process of G0-G1 transition in unperturbed and drug-treated cell populations.

Relation between carotid artery atherosclerosis and time on dialysis. A non-invasive study in vivo.

Patients undergoing maintenance hemodialysis have a high cardiovascular mortality but there are conflicting reports in the literature regarding the possibility of an accelerated atherosclerotic process in these patients. The aim of this study was to assess, in vivo, the extent of atherosclerotic lesions in the common carotid arteries and in the aorta of patients undergoing hemodialysis and their relation to clinical variables and risk factors. An ultrasonic system was employed to measure intimal-medial thickness in the carotid arteries and compliance in the aortic iliac axis. A positive significant relation emerged between carotid intimal-medial thickness and the length of time on dialysis (p < or = 0.001). Furthermore there was a significant correlation between intimal-medial thickness and LDL cholesterol (p < or = 0.001). No correlation was found between aortic-iliac compliance and the clinical variables considered. Non-invasive examination of the peripheral arteries with ultrasound could be a useful technique in the follow-up of patients on dialytic treatment; it could aid in the identification of the patients who are at increased vascular risk.

[Wall stress in the assessment of left ventricular function in surgery of abdominal aortic aneurysm. Validity and importance of transesophageal echocardiography (TEE) in intraoperative monitoring]. / Lo stress parietale nella valutazione della funzione del ventricolo sinistro nella chirurgia degli aneurismi dell'aorta addominale. Validità ed importanza dell'ecocardiografia transesofagea (TEE), nel monitoraggio intraoperatorio.

OBJECTIVE: To verify the applicability and the clinical significance of left ventricular wall stress determinations by intraoperative transesophageal echocardiography (TEE) during resections of abdominal aortic aneurysms. DESIGNS: Prospective comparison of changes in left ventricular wall stress between two groups of patients with and without coronary artery disease. SETTING: Operatory room of Universitary Institute. PATIENTS: Twenty-three patients with abdominal aortic aneurysms; 8 had clinically evident coronary artery disease (CAD+); 15 patients did not have clinical or electrocardiographic evidence of coronary artery disease (CAD-). INTERVENTIONS: Resection of the aortic aneurysm and insertion of a synthetic prosthesis. MEASUREMENTS AND MAIN RESULTS: During operation transesophageal monitoring of left ventricular volumes and wall stress was performed during induction of anesthesia (T1), for two minutes after aortic clamping (T2), at the end of the proximal anastomosis (T3), for two minutes after aortic declamping (T4) and at the end of the procedure (T5). Circumpherential stress at end systole (sES) and end diastole (sED) was more sensitive than hemodynamic and volumetric parameters in detecting changes i function of the ischemic myocardium. In detail we observed: a significant increase of sES in CAD+versus CAD- at T2: 98 (sd 18) vs 83 (sd 14) 10(3) dyne/cm2. a significant increase of sED in CAD + versus CAD- at T2: 28.5 (sd 6) vs 22 (sd 4.5) 10(3) dyne/cm3. a similar trend of sES and sED at T4: 73 (sd 20.5) vs 46 (sd 15) 10(3) dyne/cm2 and 31 (sd 12) vs 16 (sd 7.7) 10(3) dyne/cm2 respectively. a significant increase of sED in CAD + at T5 (about 20' after T4): 26.5 (sd 9.5) vs 16 (sd 5.2) 10(3) dyne/cm2 which is expression of a persistent reduction of ventricular compliance in the ischemic patients. CONCLUSIONS: Wall stress modifies MVO2 and subsequently is sensitive in detecting changes in myocardial performance. TEE could valuably integrate routine hemodynamic monitoring of patients with coronary heart disease who undergo surgical resection of abdominal aortic aneurysms.

Antimycobacterial therapy in Crohn's disease: results of a controlled, double-blind trial with a multiple antibiotic regimen.

UNLABELLED: Several recent reports have suggested an association of atypical mycobacteria with Crohn's disease. OBJECTIVES: The goal of this double-blind, placebo-controlled trial was to determine the efficacy of treatment with antimycobacterial drugs in maintaining clinical remission and in reducing active inflammatory lesions. METHODS: Forty patients (15 male) with refractory, steroid-dependent Crohn's disease were randomized to receive 2 months of tapering steroids plus either a 9-month regimen of ethambutol, clofazimine, dapsone and 1-day dose only of rifampicin (n = 22), or identical placebo. RESULTS: Three patients (two on active drug) were unable to discontinue steroids, and one patient on active drug was withdrawn for side effects during the first 2 months. Three of the remaining 19 patients on active drug relapsed during the study period, compared with 11 of 17 on placebo (log likelihood ratio = 4.6; p = 0.03). Another patient was withdrawn in remission at 5 months for anemia related to dapsone. Nine patients whose disease relapsed or persisted on placebo were crossed over to active drug; five achieved sustained remission, two failed, and two were withdrawn for side effects. Substantial endoscopic or radiologic healing did not occur. CONCLUSION: This study suggests that the treatment regimen with rifampicin, ethambutol, clofazimine, and dapsone is effective in relief of symptoms and maintenance of remission in some Crohn's disease patients.

Validation study of thoracic fluid bioimpedance for assessing the haemodialysis-induced changes in total body fluids.

Thoracic fluid bioimpedance (TFB) has been proposed as a noninvasive technique for monitoring both haemodynamics and fluid homeostasis in patients on regular dialysis. To validate the reliability of TFB in assessing the haemodialysis (HD)-induced changes in plasma volume (PV) in these patients, we examined the changes of TFB during and after HD in relation to those in total plasma protein (TP) concentration, haematocrit (Ht), heart rate (HR), total body water (TBW) and plasma angiotensin II (A-II) concentration. Data were recorded in 13 HD patients with a wide range of interdialytic weight gains before, at the end, and 4, 8, 24, 48 h after the HD session. We found that the percent TFB changes were closely and inversely related with those of TP, Ht and TBW (r = -0.54, r = -0.45 and -0.68, respectively, p < 0.001 for all). Similar relations were found between the percent changes in TFB and those in absolute TP and Ht. In addition, a direct relation was found between the percent changes of TFB and those of HR and of A-II (r = 0.33 and r = 0.31, respectively, p < 0.01 for both). These data indicate that TFB is a reliable method for evaluating the HD-induced changes of intra- and extravascular fluids and, with respect to the conventional techniques, has the advantage of providing this information dynamically, and in conjunction with the haemodynamic data of patients.

Multiparametric assessment of the cell-cycle effects of tamoxifen on mcf-7 human breast-cancer cells.

Tamoxifen (TAM)-induced changes in proliferation kinetics of the human breast cancer cell line MCF-7 were investigated using dual parameter flow cytometry (FCM) of bromodeoxyuridine (BrdU) immunolabelling and of the expression of cell-cycle related proteins (the proliferating cell nuclear antigen, PCNA, and the non proliferation-specific protein, Statin), versus the DNA content. Single-parameter FCM DNA histograms confirmed that after 96 hours of treatment with 10(-7) M TAM the fraction of S-phase cells decreased significantly, with a simultaneous accumulation of cells in the G(0)/G(1) range of DNA content. In dual-parameter FCM cytograms, the fraction of BrdU-positive cells after TAM exposure was significantly lower than in the controls, and no unlabeled S-phase cells were found. The TAM-induced block in G(0)/G(1) phase was paralleled by a decrease in the number of cells with a DNA content typical of the S-phase expressing PCNA, and by an increase in Statin-positive (G(0)) cells. Upon readdition of 10(-9) M 17 beta-estradiol (E2) to the TAM-treated cultures, BrdU-labelling as well as PCNA expression levels increased significantly, whereas the fraction of Statin-positive cells remained higher than in the controls. The results obtained confirm that the TAM-induced inhibition of cell growth is associated with major changes in the cell cycle parameters of MCF-7 cells, and provide experimental evidence that two main mechanisms are operating: the accumulation of cells in G(1), before the onset of S-phase, and the exit of some cells from the cycling compartment; however, some of those that are blocked at G(0); cannot be totally reversed by estrogens and may be permanent; These data should be taken into account in the attempt to combine the antiestrogen treatment with chemotherapy more effectively.

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.

[Color-Doppler semiology in transplanted kidney]. / Semeiotica color-Doppler nel rene trapiantato.

Color-encoded duplex ultrasonography (CEDU) makes a more accurate technique in kidney graft monitoring by combining real-time US with pulsed Doppler studies of renal vasculature. It is a non-invasive and easy technique. Suitable to study the whole renal artery and vein, CEDU also allows the qualitative and quantitative assessment of the intrarenal vasculature and therefore the easy diagnosis of such vessel dysfunctions as arteriovenous fistulas following biopsy. Moreover, Doppler spectral analysis can be used to distinguish among different causes of renal allograft dysfunction--i.e. rejection, cyclosporine nephrotoxicity or acute tubular necrosis. The value of the resistive index for the differential diagnosis is discussed. CEDU allows a more reliable measurement of renal blood flow thanks to the more precise evaluation of renal artery diameter and mean flow velocity.

[Role of echo-color Doppler in the monitoring of the transplanted kidney]. / Ruolo dell'eco-color-Doppler nel monitoraggio del rene trapiantato.

Renal allografts are subject to many vascular complications. Doppler US is a non-invasive technique that can evaluate many of the potential complications related to renal allografts, such as rejection, cyclosporine toxicity, acute tubular necrosis, renal artery stenosis, renal vein thrombosis. Changes in the resistive index (RI), which accurately reflects renal vascular impedance, have been employed in the evaluation of renal allograft dysfunction. A review of the literature shows conflicting results with values of sensitivity and specificity ranging from 0.96 and 0.97 to 0.46 and 0.80, respectively. It seems that an increase in the RI is a nonspecific sign in determining the underlying abnormality due to the unique features of the vascular bed of each allograft which results in poor correlative values for single isolated measurements of RI. Serial studies, employing a baseline value, can be useful in the follow-up of graft dysfunction.

[Effects of etozoline in patients with acute cardiac failure]. / Effetto dell'etozolina in pazienti affetti da scompenso cardiaco acuto.

Fifteen patients (10 M, 5 F; mean age 68.3 years) with acute heart failure III-IV NYHA class, were treated with etozoline (mean daily dose 666 mg) for 7 days. Symptoms of cardiac decompensation disappeared; no changes of blood pressure and heart rate were recorded. Daily urine volume increased from 790.9 +/- 327.0 ml to 1622.7 +/- 220.6 ml and body weight decreased from 71.5 +/- 5.8 kg to 69.6 +/- 5.2 kg. No changes of serum Na+, urinary Na+ excretion, appeared, but K+ excretion increased and serum K+ fell from 4.1 +/- 0.4 mEq/l to 3.6 +/- 0.45 mEq/l. No subjective side effects were reported. Renal-liver function and glyco-lipid balance were not affected. In conclusion, etozoline is a safe and effective diuretic agent in the treatment of acute cardiac failure.

Crohn's disease and mycobacteria: two cases of Crohn's disease with high anti-mycobacterial antibody levels cured by dapsone therapy.

Five patients with Crohn's colitis or ileocolitis (CD) refractory to conventional therapy were enrolled in an open trial with dapsone (100 mg/day). This therapy was apparently effective in two out of five patients. In these patients we observed a clinical improvement after one month of therapy and, in the first patient, a complete healing of all the cutaneous and rectal ulcers. In the two responders antibody levels to a soluble extract of M. paratuberculosis (MPSE) were significantly greater than in the three non-responders (P = 0.03); in the first patient, moreover, there was a rise of 39% in antibody titres following the treatment. This rise in antibody levels, that might be expected following death of the pathogen and release of antigen, is similar to that observed after treatment of tuberculosis. Our data suggest that a mycobacterial species or another pathogen that cross-react with those of MPSE, sensitive to dapsone, may in a subset of cases be responsible for the development of CD. This is the first report of clinical cure with an agent active against specific bacterial species, associated with immunologic confirmation of a response.

Influence of the heart rate on mean circumferential shortening velocity: echocardiographic study of 183 normal subjects.

Echocardiography was used to explore the influence of independent variables (age, body surface area and heart rate) on the mean circumferential shortening velocity (MVCF) in 183 healthy subjects. Multiple stepwise regression analysis shows that heart rate is the only variable of the three just mentioned that influences MVCF. A regression equation is evolved and proposed as an index of MVCF correction for varying heart rates.

[Polygraphic study of left ventricular function in severe hepatitis cirrhosis]. / Studio poligrafico della funzione ventricolare sinistra nella cirrosi epatica severa.

Systolic time intervals have been investigated in 26 subjects with severe liver cirrhosis. The patients were subdivided as follows: 15 with alcoholic cirrhosis; 11 with post-necrotic cirrhosis. Systolic time intervals were recorded and measured according to the Weissler's method. The left ventricular ejection time was significantly shortened due to the low systemic vascular resistance, while the pre-ejection period was unchanged, denoting preserved myocardial contractility. The isometric contraction time was also significantly shortened, thus confirming a good myocardial response to increased pre-load due to hyperdynamic state. The cardiac performance as assessed by systolic time intervals, is unchanged in hepatic cirrhosis.